Lox and loxl2 inhibitors and uses thereof

ABSTRACT

The present application relates to anti-LOX and anti-LOXL2 antibodies and their use in purification, diagnostic and therapeutic methods. Antibodies include monoclonal antibodies, humanized antibodies and functional fragments thereof. Anti-LOX and anti-LOXL2 antibodies can be used to identify and treat conditions such as a fibrotic condition, angiogenesis, or to prevent a transition from an epithelial cell state to a mesenchymal cell state.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/185,050, filed, Aug. 1, 2008, which application claims the benefit ofU.S. Provisional Application No. 60/963,282, entitled “Methods forSelecting Inhibitors of Tumor Invasion, Angiogenesis, and Metastasis,”filed Aug. 2, 2007; U.S. Provisional Application No. 60/963,249,entitled “Treatment of Diseases With Inhibitors of Active LysylOxidase,” filed Aug. 2, 2007; U.S. Provisional Application No.60/963,214, entitled “Treatment of Diseases Through Inhibition of BothLysyl Oxidase and Lysyl Oxidase-Like Proteins,” filed Aug. 2, 2007; U.S.Provisional Application No. 60/963,248, entitled “Diagnosis orMonitoring of Diseases by Assessing Active Lysyl Oxidase Levels orActivity,” filed Aug. 2, 2007; and U.S. Provisional Application No.60/963,246, entitled “Combination Therapy Including Lysyl OxidaseModulators,” filed Aug. 2, 2007, each of which applications isincorporated herein in its entirety by reference.

This application is related to co-pending U.S. patent application Ser.No. 12/185,054, filed Aug. 1, 2008, and PCT Patent Application SerialNo. PCT/US2008/009354, filed Aug. 1, 2008, each of which applications isincorporated herein in its entirety by reference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 246102006910SeqList.txt,date recorded: Aug. 7, 2013, size: 91,657 bytes).

BACKGROUND ART

Cancer is a serious public health problem in the United States and otherdeveloped countries. Currently, one in four deaths in the United Statesis due to cancer. Cancer therapy involves treating patients withchemotherapeutic drugs to kill tumor cells. However, subsets of tumorcells are frequently resistant to drug therapy and survive tore-populate at sites of origin and at distant metastatic sites, leadingto detectable disease recurrence and morbidity. Many carcinoma tumorcells that have the properties of increased invasive and metastaticcapacity, and altered drug resistance, are thought to have undergone amorphological transformation encompassing or similar to EMT(epithelial-mesenchymal transition). Cells undergoing EMT lose thenormal adhesive properties of epithelial cells and undergo a spectrum ofchanges including loss of E-cadherin expression and expression ofmesenchymal markers, increased motility, increased invasiveness, andincreased resistance to cell death.

The leading therapies for cancer are currently surgery, radiation andchemotherapy. Chemotherapeutic approaches such as anti-tumorantibiotics, alkylating agents, nitrosourea compounds, vinca alkaloids,steroid hormones, and anti-metabolites form the bulk of therapiesavailable to oncologists. Despite advances in the field of cancertreatment, cancer remains a major health problem.

Angiogenesis, the formation of new blood vessels out of pre-existingcapillaries, is a sequence of events that is of key importance in abroad array of physiologic and pathologic processes. Normal tissuegrowth, such as in embryonic development, wound healing, and themenstrual cycle, is characterized by dependence on new vessel formationfor the supply of oxygen and nutrients as well as removal of wasteproducts. A large number of different and unrelated diseases are alsoassociated with formation of new vasculature. Among certain pathologiesare conditions in which angiogenesis is low, and should be enhanced toimprove disease conditions. More frequently, however, excessiveangiogenesis is an important characteristic of various pathologies,including pathologies characterized or associated with an abnormal oruncontrolled proliferation of cells. Pathologies which involve excessiveangiogenesis include, for example, cancer (both solid and hematologictumors), cardiovascular diseases (such as atherosclerosis andrestenosis), chronic inflammation (rheumatoid arthritis, Crohn'sdisease), diabetes (diabetic retinopathy), psoriasis, endometriosis,neovascular glaucoma and adiposity (3). These conditions may benefitfrom chemotherapeutic inhibition of angiogenesis.

Generally speaking, the angiogenic process entails the proliferation andmigration of a normally quiescent endothelium, the controlledproteolysis of the pericellular matrix, and the synthesis of newextracellular matrix components by developing capillaries. Theestablishment of new intra- and intercellular contacts and themorphological differentiation of endothelial cells to capillary-liketubular networks provide support for their subsequent maturation,branching, remodeling and selective regression to form a highlyorganized, functional microvascular network. The autocrine, paracrineand amphicrine interactions of the vascular endothelium with itssurrounding stromal components, as well as with the pro-angiogenic andangiostatic cytokines and growth factors orchestrating physiologicangiogenesis, are normally tightly regulated both spatially andtemporally.

Angiogenesis is crucial to the growth of neoplastic tissues. For morethan 100 years, tumors have been observed to be more vascular thannormal tissues. Several experimental studies have suggested that bothprimary tumor growth and metastasis require neovascularization. Incontrast to the well orchestrated process described above for normaltissue growth, the pathologic angiogenesis necessary for active tumorgrowth is generally sustained and persistent, with the initialacquisition of the angiogenic phenotype being a common mechanism for thedevelopment of a variety of solid and hematopoietic tumor types. Tumorsthat are unable to recruit and sustain a vascular network typicallyremain dormant as asymptomatic lesions in situ. Metastasis is alsoangiogenesis-dependent: for a tumor cell to metastasize successfully, itgenerally must gain access to the vasculature in the primary tumor,survive the circulation, arrest in the microvasculature of the targetorgan, exit from this vasculature, grow in the target organ, and induceangiogenesis at the target site. Thus, angiogenesis appears to benecessary at the beginning as well as the completion of the metastaticcascade.

The criticality of angiogenesis to the growth and metastasis ofneoplasms thus provides an optimal potential target for chemotherapeuticefforts. Appropriate anti-angiogenic agents may act directly orindirectly to influence tumor-associated angiogenesis either by delayingits onset (i.e., blocking an “angiogenic switch”) or by blocking thesustained and focal neovascularization that is characteristic of manytumor types. Anti-angiogenesis therapies directed against thetumor-associated endothelium and the multiple molecular and cellularprocesses and targets implicated in sustained pathologic angiogenesisare being actively evaluated for their safety and efficacy in multipleclinical trials. However, there has been limited success to date withthe discovery and/or identification of safe and/or effectiveanti-angiogenic agents.

Fibrosis is the abnormal accumulation of fibrous tissue that can occuras a part of the wound-healing process in damaged tissue. Such tissuedamage may result from physical injury, inflammation, infection,exposure to toxins, and other causes.

Liver (hepatic) fibrosis, for example, occurs as a part of thewound-healing response to chronic liver injury. Fibrosis occurs as acomplication of haemochromatosis, Wilson's disease, alcoholism,schistosomiasis, viral hepatitis, bile duct obstruction, exposure totoxins, and metabolic disorders. This formation of scar tissue isbelieved to represent an attempt by the body to encapsulate the injuredtissue. Liver fibrosis is characterized by the accumulation ofextracellular matrix that can be distinguished qualitatively from thatin normal liver. Left unchecked, hepatic fibrosis progresses tocirrhosis (defined by the presence of encapsulated nodules), liverfailure, and death.

As summarized by Li and Friedman (Gastroenterol. Hepatol. 14:618-633,1999), actual and proposed therapeutic strategies for liver fibrosisinclude removal of the underlying cause (e.g., toxin or infectiousagent), suppression of inflammation (using, e.g., corticosteroids, IL-1receptor antagonists, or other agents), down-regulation of stellate cellactivation using, e.g., gamma interferon or antioxidants), promotion ofmatrix degradation, or promotion of stellate cell apoptosis. Despiterecent progress, many of these strategies are still in the experimentalstage, and existing therapies are aimed at suppressing inflammationrather than addressing the underlying biochemical processes. Thus, thereremains a need in the art for materials and methods for treatingfibrosis, including liver and lung fibrosis.

Fibrotic tissues accumulate in the heart and blood vessels as a resultof hypertension, hypertensive heart disease, atherosclerosis, andmyocardial infarction. High blood pressure, or hypertension, can because by a variety of factors and often leads to the development ofHypertensive Heart Disease (HHD) with progression to cardiac arrest andmyocardial infarction. Similarly, atherosclerosis and other ischemicheart diseases often also result in cardiac arrest. These cardiovasculardiseases all exhibit an accumulation of extra-cellular matrix orfibrotic deposition which results in stiffening of the vasculature andstiffening of the cardiac tissue itself. This deposition of fibroticmaterial is a response to the damage induced by the hypertensive and/orsclerotic state, but the effects of this response also result in thenegative effects of vascular and cardiac stiffening as well as ventricleenlargement. Additionally, it is believed that the increased cardiacfibrosis seen in cardiovascular disease disrupts or alters the signalstransmitted to cardiomyocytes via the tissue scaffolding of the heart,further leading to disruption of efficient cardiac function andpromoting cardiac arrest and myocardial infarction.

SUMMARY OF THE INVENTION

Epithelial-to-Mesenchymal Transition (EMT) refers to the process wherebya cell with a gene expression/phenotype characteristic of epithelialcell (i.e., expressing specific proteins, factors, and molecules)changes or alters the genes or their level of expression which resultsin a change in the phenotype of the cell as exhibited by the alterationor change in the genes expressed.

Compositions are needed which prevent EMT and which are effective inblocking the activity of enzymes such as LOX and LOXL2. Such inhibitorsare useful in treating diseases and disorders associated with aberrantlevels of LOX and LOXL2.

Antibodies that bind to enzymes can be competitive inhibitors,uncompetitive inhibitors or non-competitive inhibitors. With respect tocompetitive inhibition, an inhibitor usually bears structural similarityto substrate. Inhibition will be noticeable at low substrateconcentrations, but can be overcome at high substrate concentrations.With respect to uncompetitive inhibition, an inhibitor binds at a sitethat becomes available after substrate is bound at the active site.Inhibition will be most noticeable at high substrate concentration. Withrespect to non-competitive inhibition, an inhibitor binds at site awayfrom substrate binding site and relative inhibition will generally bethe same at all substrate concentrations. In one embodiment, an antibodyor antigen binding fragment thereof, described herein specifically bindsboth full-length and processed LOX or LOXL2. In one aspect, bothfull-length and processed LOX or LOXL2 are active forms of the enzyme.

Provided herein is an isolated antibody or antigen binding fragmentthereof, that specifically binds to an epitope having an amino acidsequence set forth as SEQ ID NO: 6. The antibody or antigen bindingfragment thereof, comprises a variable heavy chain having at least 75%amino acid sequence identity to an amino acid sequence set forth as SEQID NO: 1 and a variable light chain having at least 75% amino acidsequence identity to an amino acid sequence set forth as SEQ ID NO: 2.

Provided herein is an isolated antibody or antigen binding fragmentthereof, comprising a variable heavy chain having at least 75% aminoacid sequence identity to an amino acid sequence set forth as SEQ ID NO:1, and a variable light chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 2. In oneembodiment, an isolated antibody or antigen binding fragment thereof,comprises a variable heavy chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 1. In anotherembodiment, an isolated antibody or antigen binding fragment thereof,comprises a variable light chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 2. In yetanother embodiment, an isolated antibody or antigen binding fragmentthereof, competes with, or specifically binds to, any of the anti-LOXL2antibodies or antigen binding fragments thereof described herein forbinding to LOXL2. Antibodies or antigen binding fragments thereof canspecifically bind to LOXL2 with a binding affinity of at least 2, 5, 10,50, 100, 500 or 1000 times greater than to at least one of LOX, LOXL1,LOXL3 or LOXL4.

Provided herein are humanized anti-LOXL2 antibodies. A humanizedantibody or antigen binding fragment thereof, can specifically binds toan epitope having an amino acid sequence set forth as SEQ ID NO: 6. Inone embodiment, the humanized antibody or antigen binding fragmentthereof, comprises a variable heavy chain having at least 75% amino acidsequence identity to an amino acid sequence set forth as SEQ ID NO: 25,26, 27 or 28 and a variable light chain having at least 75% amino acidsequence identity to an amino acid sequence set forth as SEQ ID NO: 30,31 or 32.

A humanized isolated antibody or antigen binding fragment thereof, cancomprise a variable heavy chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: SEQ ID NO:25, 26, 27 or 28, and a variable light chain having at least 75% aminoacid sequence identity to an amino acid sequence set forth as SEQ ID NO:30, 31 or 32.

A humanized antibody or antigen binding fragment thereof, can comprise avariable heavy chain having at least 75% amino acid sequence identity toan amino acid sequence set forth as SEQ ID NO: 25, 26, 27 or 28.

A humanized antibody or antigen binding fragment thereof, can comprise avariable light chain having at least 75% amino acid sequence identity toan amino acid sequence set forth as SEQ ID NO: 30, 31 or 32.Combinations of variable heavy chains and variable light chains can bemade to assess binding affinity.

Provided herein is a humanized antibody or antigen binding fragmentthereof, that competes with, or specifically binds to, an antibody orantigen binding fragment thereof described herein for binding to LOXL2.

Provided herein is a humanized antibody, or antigen-binding fragmentthereof, which binds LOXL2, comprising a heavy chain variable region anda light chain variable region, wherein said heavy chain variable regioncomprises:

-   -   (i) a heavy chain FR1 having the amino acid sequence of SEQ ID        NO: 33 or the amino acid sequence of SEQ ID NO: 33 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of glutamine (Q) by valine (V) or a            conservative substitution thereof at position 24;        -   (b) a substitution of leucine (L) by valine (V) or a            conservative substitution thereof at position 30;        -   (c) a substitution of valine (V) by lysine (K) or a            conservative substitution thereof at position 31;        -   (d) a substitution of arginine (R) by lysine (K) or a            conservative substitution thereof at position 32; and        -   (e) a substitution of threonine (T) by alanine (A) or a            conservative substitution thereof at position 35;        -   and a deletion of amino acid residues 1-19;    -   (ii) a heavy chain FR2 having the amino acid sequence of SEQ ID        NO: 34 or the amino acid sequence of SEQ ID NO: 34 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of lysine (K) by arginine (R) or a            conservative substitution thereof at position 3;        -   (b) a substitution of arginine (R) by alanine (A) or a            conservative substitution thereof at position 5, and    -   (iii) a heavy chain FR3 having the amino acid sequence of SEQ ID        NO: 35 or the amino acid sequence of SEQ ID NO: 35 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of lysine (K) by arginine (R) or a            conservative substitution thereof at position 1;        -   (b) a substitution of alanine (A) by valine (V) or a            conservative substitution thereof at position 2;        -   (c) a substitution of leucine (L) by isoleucine (I) or a            conservative substitution thereof at position 4;        -   (d) a substitution of serine (S) by threonine (T) or a            conservative substitution thereof at position 10;        -   (e) a substitution of glutamine (Q) by glutamic acid (E) or            a conservative substitution thereof at position 16;        -   (f) a substitution of threonine (T) by arginine (R) or a            conservative substitution thereof at position 21;        -   (g) a substitution of aspartic acid (D) by glutamic acid (E)            or a conservative substitution thereof at position 23;        -   (h) a substitution of serine (S) by threonine (T) or a            conservative substitution thereof at position 25; and a            substitution of phenylalanine (F) by tyrosine (Y) or a            conservative substitution thereof at position 29;        -   and    -   (iv) a heavy chain FR4 having the amino acid sequence of SEQ ID        NO: 36 or the amino acid sequence of SEQ ID NO: 36 but for a        substitution of lysine (K) by valine (V) or a conservative        substitution thereof at position 7,    -   and wherein said light chain variable region comprises:    -   (i) a light chain FR1 having the amino acid sequence of SEQ ID        NO: 49 or the amino acid sequence of SEQ ID NO: 49 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of alanine (A) by threonine (T) or a            conservative substitution thereof at position 27;        -   (b) a substitution of alanine (A) by proline (P) or a            conservative substitution thereof at position 28;        -   (c) a substitution of proline (P) by leucine (L) or a            conservative substitution thereof at position 29;        -   (d) a substitution of valine (V) by leucine (L) or a            conservative substitution thereof at position 31;        -   (e) a substitution of glutamic acid (E) by glutamine (Q) or            a conservative substitution thereof at position 37;        -   (d) a substitution of serine (S) by proline (P) or a            conservative substitution thereof at position 38;        -   (f) a substitution of valine (V) by alanine (A) or a            conservative substitution thereof at position 39;        -   and a deletion of amino acid residues 1-20;    -   (ii) a light chain FR2 having the amino acid sequence of SEQ ID        NO: 50 or the amino acid sequence of SEQ ID NO: 50 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of phenylalanine (F) by tyrosine (Y) or a            conservative substitution thereof at position 2; and        -   (b) a substitution of arginine (R) by lysine (K) or a            conservative substitution thereof at position 5;    -   (iii) a light chain FR3 having the amino acid sequence of SEQ ID        NO: 51 or the amino acid sequence of SEQ ID NO: 51 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of alanine (A) by aspartic acid (D) or a            conservative substitution thereof at position 14; and        -   (b) a substitution of arginine (R) by lysine (K) or a            conservative substitution thereof at position 18;    -   and    -   (iv) a light chain FR4 having the amino acid sequence of SEQ ID        NO: 52 or the amino acid sequence of SEQ ID NO: 52 but for a        substitution of leucine (L) by valine (V) or a conservative        substitution thereof at position 7;

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region FR1 having an amino acidsequence as set forth in SEQ ID NO: 33, 37 or 44; a heavy chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 34,38 or 45; a heavy chain variable region FR3 having an amino acidsequence as set forth in SEQ ID NO: 35, 39, 46, 47 or 48; a heavy chainvariable region FR4 having an amino acid sequence as set forth in SEQ IDNO: 36 or 40; a light chain variable region FR1 having an amino acidsequence as set forth in SEQ ID NO: 49 or 53; a light chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 50,54 or 60; a light chain variable region FR3 having an amino acidsequence as set forth in SEQ ID NO: 51, 55 or 61; and a light chainvariable region FR4 having an amino acid sequence as set forth in SEQ IDNO: 52 or 56.

Provided herein are antibodies that specifically bind to LOX. In oneaspect, an isolated antibody or antigen binding fragment thereof, cancomprise a variable heavy chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 3 and avariable light chain having at least 75% amino acid sequence identity toan amino acid sequence set forth as SEQ ID NO: 4 or 5. In anotheraspect, an isolated antibody or antigen binding fragment thereof, cancomprise a variable heavy chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 3. In yetanother aspect, an isolated antibody or antigen binding fragmentthereof, can comprise a variable light chain having at least 75% aminoacid sequence identity to an amino acid sequence set forth as SEQ ID NO:4 or 5.

Provided herein is an isolated antibody or antigen binding fragmentthereof, that competes with, or specifically binds to, an antibody orantigen binding fragment thereof of any one of the anti-LOX antibodiesdescribed herein for binding to LOX.

An anti-LOX antibody can specifically bind to LOX with a bindingaffinity of at least 2, 5, 10, 50, 100, 500 or 1000 times greater thanto at least one of LOXL1, LOXL2, LOXL3 or LOXL4.

An isolated antibody or antigen binding fragment thereof can be labeledwith a detectable label, a therapeutic label or both.

In one embodiment, an antigen binding fragment is, for example, avariable heavy chain, a variable light chain, a Fv, a scFv, a Fab, aF(ab′)2, a genetically engineered antibody, a monoclonal antibody, or ahumanized antibody.

Provided herein is a kit for treating a condition associated with LOX orLOXL2, comprising a composition of an antibody or antigen bindingfragment thereof of any one of the preceding embodiments and apharmaceutically acceptable carrier or excipient. A condition associatedwith LOX or LOXL2 can be, for example, a tumor, a metastasis,angiogenesis, or fibrosis. A kit can further comprise a detectablelabel, a therapeutic label or both. Kits can further comprise writteninstructions describing how to conjugate the antibody or antigen bindingfragment thereof with the detectable label, a therapeutic label or both.Furthermore, written instructions can describe how to administer theantibody or antigen binding fragment thereof. In one embodiment,compositions in the kit are free of pyrogens and can, in some instances,is lyophilized.

Provided herein is a method of diagnosing a condition associated withLOX or LOXL2 comprising assessing a level of LOX and/or LOXL2 in asample of a subject by contacting said sample with an antibody orantigen binding fragment thereof described herein, wherein a change inlevel of LOX and/or LOXL2 in the sample in comparison with a referencesample indicates the presence or increase of a tumor or metastasis. Acondition associated with LOX or LOXL2 can be, for example, a tumor, ametastasis, angiogenesis, or a fibrotic condition. In one embodiment, anincrease in LOX and/or LOXL2 levels in the sample in comparison with areference sample indicates the presence of a tumor or metastasis or anincrease in tumor or metastatic growth. A reference sample is a sampletaken from the subject at an earlier time point or a sample from anotherindividual. Levels of LOX and/or LOXL2 levels in the sample are detectedby contacting the sample with any of the antibodies or antigen bindingfragments thereof described herein. For detection purposes, an antibodyor antigen binding fragment thereof is detectably labeled as neededdepending upon the method used to assess binding.

Provided herein is a method of inhibiting LOXL2 by contacting a sampleor a cellular tissue with an antibody or antigen binding fragmentthereof, described herein. In one embodiment, binding of said antibodyor antigen binding fragment thereof to LOXL2 inhibits enzymatic activityof LOXL2.

Provided herein is a method of inhibiting LOX by contacting a sample orcellular tissue with an antibody or antigen binding fragment thereof,described herein. In one embodiment, binding of said antibody or antigenbinding fragment thereof to LOX inhibits enzymatic activity of LOX.Contacting can occur in vitro, in vivo or ex vivo. Inhibiting LOX orLOXL2 can reduce tumor growth in a subject either partially orcompletely. Inhibiting LOX or LOXL2 can reduce angiogenesis in a subjectsuch that a therapeutic benefit occurs. Inhibiting LOX or LOXL2 canreduce fibrosis in a subject such that a therapeutic benefit occurs.

Provided herein is a method of reducing growth of a tumor in a subject,comprising administering an antibody or antigen binding fragmentthereof, described herein. A tumor can be a primary tumor or ametastatic tumor. In one aspect, a tumor is, for example, Lung cancer(including lung adenocarcinoma, squamous cell carcinoma, large cellcarcinoma, bronchioloalveolar carcinoma, non-small-cell carcinoma, smallcell carcinoma, mesothelioma); breast cancer (including ductalcarcinoma, lobular carcinoma, inflammatory breast cancer, clear cellcarcinoma, mucinous carcinoma,); colorectal cancer (colon cancer, rectalcancer); anal cancer; pancreatic cancer (including pancreaticadenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostatecancer; ovarian carcinoma (ovarian epithelial carcinoma or surfaceepithelial-stromal tumour including serous tumour, endometrioid tumorand mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bileduct carcinoma (including hepatocelluar carcinoma, cholangiocarcinoma,hemangioma); esophageal carcinoma (including esophageal adenocarcinomaand squamous cell carcinoma); non-Hodgkin's lymphoma; bladder carcinoma;carcinoma of the uterus (including endometrial adenocarcinoma, uterinepapillary serous carcinoma, uterine clear-cell carcinoma, uterinesarcomas and leiomyosarcomas, mixed mullerian tumors); glioma,glioblastoma, medullablastoma, and other tumors of the brain; kidneycancers (including renal cell carcinoma, clear cell carcinoma, Wilm'stumor); cancer of the head and neck (including squamous cellcarcinomas); cancer of the stomach (stomach adenocarcinoma,gastrointestinal stromal tumor); multiple myeloma; testicular cancer;germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids ofthe gastrointestinal tract, breast, and other organs; signet ring cellcarcinoma; mesenchymal tumors including sarcomas, fibrosarcomas,haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatousstromal hyperplasia, myofibroblastoma, fibromatosis, inflammatorymyofibroblastic tumour, lipoma, angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma,osteosarcoma, leiomyoma or a leiomysarcoma. In one embodiment, a tumoris, for example, a colon tumor, an ovarian tumor, a lung tumor, anesophageal tumor, a breast tumor, a prostate tumor, a carcinoma. Tumorsize in the subject can be reduced by at least 10%, 25%, 50%, 70%, 90%,95%, or more following treatment as compared to the tumor in the subjectprior to treatment. In one aspect, the survival of a subject with atumor is increased by at least 10 days, 1 month, 3 months, 6 months, 1year, 2 years, 5 years, 10 years, or more compared to a subject that isnot administered the antibody or antigen binding fragment thereof.Metastatic tumor burden of a subject can be stabilized followingadministration of an antibody or antigen binding fragment thereof,described herein. For instance, metastatic tumor burden can bestabilized for at least 10 days, 1 month, 3 months, 6 months, 1 year, 2years, 5 years, 10 years or more.

An antibody or antigen-binding fragment thereof described herein canspecifically bind to a secreted or mature form of hLOX but not to apreproprotein of hLOX having an amino acid sequence of SEQ ID NO: 7. Inone embodiment, secreted form of hLOX has an amino acid sequence of SEQID NO: 8, 62 or 63. In one embodiment, the mature form of hLOX has anamino acid sequence of SEQ ID NO: 9.

Provided herein is a method of inhibiting angiogenesis in a subject byan antibody or antigen binding fragment thereof, described herein.

Provided herein is a method of inhibiting a fibrotic disease in asubject by administering an antibody or antigen binding fragmentthereof, described herein. Fibrotic diseases include, but are notlimited to, liver fibrosis, lung fibrosis, kidney fibrosis, cardiacfibrosis and schleroderma. In one embodiment, kidney fibrosis includes,but is not limited to, diabetic nephropathy, vesicoureteral reflux,tubulointerstitial renal fibrosis; glomerulonephritis or glomerularnephritis, including focal segmental glomerulosclerosis and membranousglomerulonephritis, and Mesangiocapillary glomerular nephritis. In oneembodiment, liver fibrosis results in cirrhosis, and associatedconditions such as chronic viral hepatitis, non-alcoholic fatty liverdisease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholicsteatohepatitis (NASH), primary biliary cirrhosis (PBC), biliarycirrhosis, and autoimmune hepatitis.

Provided herein is a method of decreasing extracellular matrix formationby contacting a sample or cellular tissue with an antibody or antigenbinding fragment thereof, described herein. Administration or contactingcan occur, in one example, by parenteral administration.

Provided herein is a method of monitoring a subject's response toadministration of an antibody or antigen binding fragment thereof,described herein by detecting by detecting LOX and/or LOXL levels and/oractivity.

In one embodiment, said antibody or antigen binding fragment thereof, islabeled with a therapeutic label.

Contemplated herein is combination therapy in which the methods furthercomprise co-administering a second therapeutic agent. In one embodiment,the second therapeutic agent is an antibody or a chemotherapeutic agent.

Provided herein is a use of an antibody or antigen binding fragmentthereof, described herein in the preparation of a formulation forinhibiting LOXL2 or LOX, reducing tumor growth, inhibiting angiogenesis,inhibiting a fibrotic disease or decreasing extracellular matrixformation in a subject. In one embodiment, said antibody or antigenbinding fragment thereof is labeled with a therapeutic label and,optionally, a diagnostic label.

Provided herein is a use of an antibody or antigen binding fragmentthereof, described herein in the preparation of a formulation fordiagnosing a tumor or metastasis comprising assessing LOX and/or LOXL2levels in a sample of a patient, wherein a change in LOX and/or LOXL2levels in the sample in comparison with a reference sample indicates thepresence of a tumor or metastasis or an increase in tumor or metastaticgrowth. In one embodiment, said antibody or antigen binding fragmentthereof is labeled with a diagnostic label.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings.

FIG. 1 illustrates Lysyl Oxidase Enzymology. LOX/L enzymes act via aping-pong mechanism which can be described by Michaelis-Menten kinetics.

FIG. 2 illustrates common modes of enzymatic inhibition.

FIG. 3 illustrates βAPN is a competitive inhibitor of LOXL2.

FIG. 4 illustrates modes of enzymatic inhibition: LOXL2.

FIG. 5 illustrates extracellular LOXL2 localization and function ofextracellular LOXL2.

FIG. 6A provides a sequence (SEQ ID NO:71) containing the amino acidsequences of a heavy chain variable region (SEQ ID NO:1) and FIG. 6Bprovides a sequence (SEQ ID NO:72) containing the amino acid sequencesof a light chain variable region (SEQ ID NO:2) of an antibody that bindsto the SRCR3-4 region of LOXL2. For each variable region, signalpeptides are shown in italics, CDRs are underlined and the beginning ofthe constant framework is shown in bold font.

FIG. 7A provides a heavy chain variable region amino acid sequence (SEQID NO:73) and FIGS. 7B and 7C provide amino acid sequences (SEQ IDNOS:4-5) of two light chain variable regions of antibodies that bind toLOX. For each variable region, signal peptides are shown in italics andCDRs are underlined.

FIG. 8 provides a protein screen B update using anti-LOXL2 antibodies.LOXL2 enzymatic activity was assessed.

FIG. 9 illustrates enzymatic activity of anti-LOXL2 antibody AB0023.

FIG. 10 demonstrates that anti-LOXL2 antibody AB0023 is anon-competitive inhibitor.

FIG. 11 illustrates the binding affinity and off-rate of anti-LOXL2antibody AB0023.

FIG. 12 illustrates anti-LOXL2 antibody AB0023 domain mapping; AB0023binds the SRCR 3-4 domain of LOXL2.

FIG. 13 shows that anti-LOXL2 antibody AB0023 demonstrates consistentinhibition of migration/invasion in collagen I and collagen IV, fromsupernatants through 10 ml prep material and scaled-up 100 ml prep andascites material. Partial inhibition also observed in cell adhesionassays. In test samples, cells in the assay migrate toward serum andfluorescence is measured to determine cell count and migration. The farleft bar is a control sample in which no antibody is present and thebottom layer contains serum (positive control for cell invasion). Thesecond bar from left is a negative control in which no antibody ispresent and the bottom layer does not contain serum.

FIG. 14 provides amino acid sequences of the variable heavy (VH) (SEQ IDNO:24) and variable light (VL) (SEQ ID NO:29) chains of murinemonoclonal antibody AB0023 (anti-hLOXL2). Complementarity determiningregions (CDRs) (SEQ ID NOS: 41-42, 70 AND 57-59, respectively, in orderof appearance) are shown by bold underlining. FIG. 14 also provides fourhumanized variants of the murine monoclonal antibody. Residues in theframework (FR) regions of the humanized variable heavy (SEQ ID NOS:25-28, respectively, in order of appearance) and light (SEQ ID NOS:30-32, respectively, in order of appearance) chains that differ from themurine monoclonal antibody are shown by dash marks (---) or byitalicized underlining.

FIG. 15 demonstrates that M64 binds to LOX in a dose dependent manner.Batch 3 has a K_(D) of 6.6 nM, Batch 4 has a K_(D) of 5.0 nM and Batch 5has a K_(D) of 5.7 nM.

FIG. 16 illustrates the binding affinity of anti-LOX antibody M64.

FIG. 17 demonstrates that anti-LOXL2 antibodies inhibited cell growth offour cancer derived cell lines.

FIG. 18 demonstrates a synergistic effect of an anti-LOX antibody incombination with cisplatin. IC50 values of M64 were also determined onfour cell lines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the field of medicine, including cancerdiagnosis and treatment. One aspect of the invention relates to LOX andLOXL2 as indicators of disease progression and a target for therapeuticagents.

The present invention provides innovative methodology and relatedcomposition and kits for diagnosing or monitoring various diseasesassociated with abnormal cell proliferation, angiogenesis and fibrosis,by using agents that specifically recognize active or mature forms oflysyl oxidase (LOX) or lysyl oxidase-like (LOXL) proteins.

Methods are provided for diagnosing or monitoring cancer metastasis in asubject, comprising: assessing active LOX or LOXL2 levels or activity inthe blood or in a tumor, whereby a change in active LOX or LOXL2 levelsor activity in the blood or in the tumor in comparison with a referencesample, indicates the presence of metastatic tumor growth.

As described in more detail below, levels of active LOX or LOXL2 can beassessed by various methods including but are not limited toimmunohistochemistry by using antibodies that specifically bind to theactive or mature form of LOX or LOXL2. Enzymatic activity of active LOXor LOXL2 can be measured by using various methods including but notlimited to chromogenic and fluorometric assays.

Also provided herein are antibodies or antigen-binding fragmentsthereof, that specifically recognize active forms of LOX or LOXL2,methods for generating antibodies against active forms of LOX or LOXL2,and method of using the antibodies to treat abnormal cell proliferation,angiogenesis and fibrosis.

I. GENERAL DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference. The headingsprovided herein are for convenience only and do not limit the inventionin any way.

As used herein, “a” or “an” means “at least one” or “one or more.”

The phrase “conservative amino acid substitution” refers to grouping ofamino acids on the basis of certain common properties. A functional wayto define common properties between individual amino acids is to analyzethe normalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schirmer,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). Examples of amino acid groups defined in this mannerinclude:

-   -   (i) a charged group, consisting of Glu and Asp, Lys, Arg and        His,    -   (ii) a positively-charged group, consisting of Lys, Arg and His,    -   (iii) a negatively-charged group, consisting of Glu and Asp,    -   (iv) an aromatic group, consisting of Phe, Tyr and Trp,    -   (v) a nitrogen ring group, consisting of His and Trp,    -   (vi) a large aliphatic non-polar group, consisting of Val, Leu        and Ile,    -   (vii) a slightly-polar group, consisting of Met and Cys,    -   (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn,        Gly, Ala, Glu, Gln and Pro,    -   (ix) an aliphatic group consisting of Val, Leu, Ile, Met and        Cys, and    -   (x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue mayform its own group, and the group formed by an individual amino acid maybe referred to simply by the one and/or three letter abbreviation forthat amino acid commonly used in the art as described above.

A “conserved residue” is an amino acid that is relatively invariantacross a range of similar proteins. Often conserved residues will varyonly by being replaced with a similar amino acid, as described above for“conservative amino acid substitution”.

The letter “x” or “xaa” as used in amino acid sequences herein isintended to indicate that any of the twenty standard amino acids may beplaced at this position unless specifically noted otherwise. For thepurposes of peptidomimetic design, an “x” or an “xaa” in an amino acidsequence may be replaced by a mimic of the amino acid present in thetarget sequence, or the amino acid may be replaced by a spacer ofessentially any form that does not interfere with the activity of thepeptidomimetic.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 40% identity, though preferably lessthan 25% identity with a sequence of the present invention. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid (nucleotide,oligonucleotide) and amino acid (protein) sequences of the presentinvention may be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members, relatedsequences or homologs. Such searches can be performed using the NBLASTand XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST amino acidsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and BLAST) can be used (see, ncbi.nlm nih gov).

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Watermanalgorithm may also be used to determine identity.

The term “substantially identical” means identity between a first aminoacid sequence that contains a sufficient or minimum number of amino acidresidues that are i) identical to, or ii) conservative substitutions ofaligned amino acid residues in a second amino acid sequence such thatthe first and second amino acid sequences can have a common structuraldomain and/or common functional activity. For example, amino acidsequences substantially identical to LOX contain a common structuraldomain having at least about 60%, or 65% identity, likely 75% identity,more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to LOX. For example, amino acid sequences that contain a commonstructural domain having at least about 60%, or 65% identity, likely 75%identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identity to LOXL2 are termed sufficiently or substantiallyidentical. In the context of nucleotide sequence, the term“substantially identical” is used herein to refer to a first nucleicacid sequence that contains a sufficient or minimum number ofnucleotides that are identical to aligned nucleotides in a secondnucleic acid sequence such that the first and second nucleotidesequences encode a polypeptide having common functional activity, orencode a common structural polypeptide domain or a common functionalpolypeptide activity. For example, nucleotide sequences having at leastabout 60%, or 65% identity, likely 75% identity, more likely 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleic acidsequences provided herein are termed substantially identical.

II. LYSYL OXIDASE (LOX) AND LYSYL OXIDASE-LIKE (LOXL) PROTEINS

Typically, solid tumors contain areas of low oxygen tension (hypoxia).Hypoxic cells present a great problem in the treatment of cancer becausethese cells are highly aggressive, metastatic and resistant to therapy.The underlying mechanisms contributing to these features are poorlyunderstood. Metastasis poses a particular problem in breast cancerbecause there is no effective treatment for the majority of patientswith detectable metastatic breast cancer (Steeg, P S. Br. Can. Res.2(6): 396-9 (2000)).

The extracellular matrix (ECM) can have a major influence on tumor cells(Chang and Werb. Trends Cell. Biol. 11: S37-43 (2001); and Radisky etal. Semin Cancer Bio. 11: 87-95 (2001)). Mice exposed to hypoxia exhibittissue specific increases in lysyl oxidase (LOX) activity, an amineoxidase that plays an essential role in the formation and maintenance ofthe ECM (Brody et al. Am. Rev. Respir. Dis. 120: 1289-95 (2001)). Arecent microarray study confirmed LOX to be a hypoxia-induced gene in avariety of cell lines (Denko, N C. Oncogene 22: 5907-14 (2003)).However, a biological role of LOX under hypoxic conditions was notidentified. LOX initiates the covalent cross-linking of collagens andelastin in the ECM, increasing insoluble matrix deposition and tensilestrength (Kagan and Li. J. Cell. Biochem. 88: 660-72 (2003)). LOXexpression is essential for wound healing and normal connective tissuefunction, and knock-out mice die soon after parturition due tocardiovascular instability (Hornstra et al. J. Biol. Chem. 278: 14387-93(2003)). Decreased LOX activity is associated with diseases such asEhler-Danlos syndrome (Pinnell, S R. J. Invest. Dermatol. 79(Supp 1):90S-92S (1982); Royce et al. Biochem. J. 192: 579-86 (1980); and Khakooet al. Clin. Genet. 51: 109-14 (1997)). Increased LOX activitycontributes to fibrotic and tissue remodeling diseases, such as livercirrhosis (Kagan, H M. Pathol. Res. Pract. 190: 910-0 (1994); Chanki etal. Br. J. Dermatol. 133: 710-5 (1995); and Ooshima and Midorikawa. Jpn.Circ. J. 41: 1337-40 (1977)).

Elevated expression of LOX correlates with increased staging in renalcell cancer (Stassar et al. Br. J. Cancer, 85: 1372-82 (2001)), andincreased LOX expression is observed in highly metastatic and/orinvasive breast cancer cell lines (Kirschmann et al. Breast Cancer Res.Treat. 55: 127-36 (1999); and Kirschmann et al. Cancer Res. 62: 4478-83(2002)). In contrast, LOX acts as a tumor suppressor in non-tumorgenicrevertants of ras-transformed fibroblasts (Smith-Mungo and Kagan. MatrixBiol. 16: 387-98 (1998)). Loss of LOX is associated with tumorigenesisin several cancer types such as gastric, colon and prostate cancers (Renet al. Cancer Res. 58: 1285-90 (1998); Cxiszar et al. Int. J. Cancer 97:636-42 (2002); and Kaneda et al. Cancer Res. 64: 6410-5 (2004)). Itwould, thus, seem that LOX's tumor suppressive role depends on cell typeand transformation status. The propeptide domain (and not the activeenzyme) was recently shown to be responsible for the tumor suppressoractivities. In breast cancer, increased LOX expression is associatedwith the early stromal reaction (Decitre et al. Lab. Invest. 78: 143-51(1998)), and treatment with antisense LOX in this cancer cell typeprevents in vitro invasion (Kirschmann et al. Cancer Res. 62: 4478-83(2002)).

The amino acid sequence of LOXL2 shares extensive sequence homology withthe conserved copper-binding and catalytic domains of both LOX and LOXL.These conserved domains are encoded by five consecutive exons within theLOX, LOXL, and LOXL2 genes that also maintain exon-intron structureconservation. Conservation of the nucleotide and deduced amino acidsequence within the carboxyl-terminal end of LOXL2, LOX, and LOXLinclude the copper-binding domain (WEWHSCHQHYH (SEQ ID NO: 66)) in LOXand LOXL and WIWHDCHRHYH (SEQ ID NO: 67) in LOXL2 with the fourhistidines that supply the nitrogen ligands for the copper coordinationcomplex specific for lysyl oxidase proteins (Krebs and Krawetz, Biochim.Biophys. Acta 1202: 7-12 (1993)). The active site in LOX(DIDCQWVDITDVPPPGDY (SEQ ID NO: 68)) and in LOXL2 (DIDCQWVDITDVPPPGDY(SEQ ID NO: 69)) contains, in each, a Tyr residue (Y) at theCOOH-terminal end, which participates together with a Lys residue in theformation of the quinone co-factor that is present in these proteins.Ten cysteines characteristic of LOX and LOXL are similarly conserved inLOXL2 (Kagan et al., (1994) in Molecular Biology and Pathology ofElastic Tissue (Mecham, R. P., and Roberts, L., eds), Ciba FoundationSymposium Series, Wiley, Chichester, UK). A growth factor and cytokinereceptor domain present in the LOX and LOXL proteins has also beenidentified within the LOXL2-derived amino acid sequence. Four repeats ofthe scavenger receptor cysteine-rich domain are also present of LOXL2(Saito et al., J. Biol. Chem. 272: 8157-8160 (1997), Resnick et al.,Trends Biochem. Sci. 19: 5-8 (1994)).

Three major transcription termination sites have been noted within3′-UTR domains of LOXL2 cDNA. The first termination site is 690 bp 3′ ofthe termination codon, the second site is 740 bp, and the finaltranscription termination site is 900 bp 3′ of the termination codon.These mRNAs all have 3′-UTRs differing slightly in size. Mostexon-intron boundaries of the LOXL2 gene show the consensus sequence(C/T)AG-exon-GT(A/G). The sizes of the 11 exons of the LOXL2 gene rangefrom 112 to 940 bp. Although the LOXL2 gene has 11 exons, fiveconsecutive exons (exons 6-10), which encode the copper-binding andcatalytic domains, exhibit 84% sequence similarity, and exon sizes arevery similar to the corresponding exons of the LOX and LOXL genes. Allthe other exons in the LOXL2 gene are divergent in both sequence andsize. LOXL2 has been identified in all tissues with the exception ofblood leukocytes. LOXL2 mRNA has been detected in heart, liver, andpancreas; expression is significantly higher in placenta, prostate,uterus, and pancreas (ratios between 2 and 3) compared with lowerexpression in brain, lung, skeletal muscle, thymus, and kidney (ratiosbelow 0.5). (Jourdan-Le Saux, et al. J. Biol. Chem., 274(18):12939-12944 (1999)).

The expression of LOX and the different LOXL proteins varies indifferent diseases. This may be due to a number of reasons, such as thedifference in tissue distribution, processing, domains, regulation ofactivity, as well as other differences between the proteins. Forexample, LOX and LOXL are implicated in fibrotic diseases as both LOXand LOXL are highly expressed in myo-fibroblasts around fibrotic areas(Kagen, Pathol. Res. Pract. 190:910-919 (1994); Murawaki et al.,Hepatology 14:1167-1173 (1991); Siegel et al., Proc. Natl. Acad. Sci.USA 75:2945-2949 (1978); Jourdan Le-Saux et al., Biochem. Biophys. Res.Comm 199:587-592 (1994); Kim et al., J. Cell Biochem. 72:181-188(1999)). LOX and the various LOXL are also implicated in a number ofcancers. For example, LOXL and LOXL4 have been shown to beepigenetically silenced and can inhibit ras/extracellularsignal-regulated kinase signaling pathway in human bladder cancer (Wu etal., Cancer Res. 67:4123-4129 (2007)). Others have shown selectiveupregulation and amplification of the LOXL4 gene in head and necksquamous cell carcinoma (Gorough et al., J. Pathol. 212:74-82 (2007)).LOX and LOXL2 have also been implicated in a number of tumors, such ascolon and esophageal cancers (Csiszar, Prog. Nucl. Acid Res. 70:1-32(2001)). In breast cancer, LOX and the LOXL family members have beenlinked to the cancer (Kirschmann et al., Cancer Res. 62:448-4483(2002)).

Lysyl oxidase catalyzes oxidative deamination of peptidyl lysine andhydroxylysine residues in collagens, and peptidyl lysine residues inelastin. The resulting peptidyl aldehydes spontaneously condense andundergo oxidation reactions to form the lysine-derived covalentcross-links required for the normal structural integrity of theextracellular matrix. In the reaction of lysyl oxidase with itssubstrates, hydrogen peroxide (H2O2) and ammonium are released inquantities stoichiometric with the peptidyl aldehyde product. See, e.g.,Kagan et al., J. Cell. Biochem. 88:660-72 (2003).

Lysyl oxidase is secreted into the extracellular environment where it isthen processed by proteolytic cleavage to a functional 30 kDa enzyme andan 18 kDa propeptide. The 30 kDa lysyl oxidase is enzymatically activewhereas the 50 kDa proenzyme is not. Procollagen C-proteinases processpro-lysyl oxidase to its active form and are products of the Bmp1, Tll1and Tll2 genes. The localization of the enzyme is mainly extracellular,although processed lysyl oxidase also localizes intracellularly andnuclearly. Sequence coding for the propeptide is moderately (60-70%)conserved among LOX and the LOXL proteins, whereas the sequence codingfor the C-terminal 30 kDa region of the proenzyme in which the activesite is located is highly conserved (approximately 95%). See Kagan etal., J. Cell Biochem. 59:329-38 (1995). LOX is induced by a number ofgrowth factors and steroids such as TGF-β, TNF-α and interferon(Csiszar, Prog. Nucl. Acid Res. 70:1-32 (2001)).

Five different lysyl oxidases are known to exist in both humans andmice, LOX and four LOX related, or LOX-like proteins (LOXL, LOXL2,LOXL3, LOXL4). LOX and the LOX-like proteins are referred tocollectively as “LOX/LOXL” for the purposes of the present disclosure.The five forms of lysyl oxidases reside on five different chromosomes.These family members show some overlap in structure and function, butappear to have distinct functions as well. For example, although themain activity of LOX is the oxidation of specific lysine residues incollagen and elastin outside of the cell, it may also actintracellularly, where it may regulate gene expression. In addition, LOXinduces chemotaxis of monocytes, fibroblasts and smooth muscle cells.Further, a deletion of LOX in knockout mice appears to be lethal atparturition (Hornstra et al., J. Biol. Chem. 278:14387-14393 (2003)),whereas LOXL deficiency causes no severe developmental phenotype(Bronson et al., Neurosci. Lett. 390:118-122 (2005)).

The main activity of LOX is the oxidation of specific lysine residues incollagen and elastin outside of the cell, however, it may also actintracellularly, where it may regulate gene expression (Li et al., Proc.Natl. Acad. Sci. USA 94:12817-12822 (1997), Giampuzzi et al., J. Biol.Chem. 275:36341-36349 (2000)) In addition, LOX induces chemotaxis ofmonocytes, fibroblasts and smooth muscle cells (Lazarus et al., MatrixBiol. 14:727-731 (1995) Nelson et al., Proc. Soc. Exp. Biol. Med.188:346-352 (1988)). LOX itself is induced by a number of growth factorsand steroids such as TGF-β, TNF-α and interferon (Csiszar, Prog. Nucl.Acid Res. 70:1-32 (2001)). Recent studies have attributed other roles toLOX in diverse biological functions such as developmental regulation,tumor suppression, cell motility, and cellular senescence. The diverserole of LOX, and its recently discovered amino oxidase family, LOX-like(LOXL), may play important roles with their intracellular andextracellular localization.

As used herein, the term “lysyl oxidase” refers to an enzyme thatcatalyzes the following reaction:peptidyl-L-lysyl-peptide+O2+H2O→peptidyl-allysyl-peptide+NH3+H2O2. Othersynonyms for lysyl oxidase (EC 1.4.3.13) include protein-lysine6-oxidase and protein-L-lysine:oxygen 6-oxidoreductase (deaminating).See, e.g., Harris et al., Biochim Biophys. Acta 341:332-44 (1974);Rayton et al., J. Biol. Chem. 254:621-26 (1979); Stassen, Biophys. Acta438:49-60 (1976). A copper-containing quinoprotein with a lysyl adductof tyrosyl quinone at its active center, LOX catalyzes the oxidation ofpeptidyl lysine to result in the formation of peptidylalpha-aminoadipic-delta-semialdehyde. Once formed, this semialdehyde canspontaneously condense with neighboring aldehydes or with other lysylgroups to from intra-and interchain cross-links. See, e.g., Rucker etal., Am. J. Clin. Nutr. 67:996S-1002S (1998).

The term “LOX” refers to an enzyme having an amino acid sequencesubstantially identical to a polypeptide expressed or translated fromone of the following sequences: EMBL/GenBank accession numbers: M94054(SEQ ID NO: 10); AAA59525.1 (SEQ ID NO: 11)-mRNA; 545875 (SEQ ID NO:12); AAB23549.1 (SEQ ID NO: 13)-mRNA; 578694 (SEQ ID NO: 14); AAB21243.1(SEQ ID NO: 15)-mRNA; AF039291 (SEQ ID NO: 16); AAD02130.1 (SEQ ID NO:17)-mRNA; BC074820 (SEQ ID NO: 18); AAH74820.1 (SEQ ID NO: 19)-mRNA;BC074872 (SEQ ID NO: 20); AAH74872.1 (SEQ ID NO: 21)-mRNA; M84150 (SEQID NO: 22); AAA59541.1 (SEQ ID NO: 23)-Genomic DNA. One embodiment ofLOX is human lysyl oxidase (hLOX) preproprotein having an amino acidsequence (SEQ ID NO: 7), a secreted hLOX after cleavage of the signalpeptide such as SEQ ID NO: 8 or a mature hLOX after proteolyticprocessing such as SEQ ID NO: 9.

LOX has highly conserved protein domains, conserved in several speciesincluding human, mouse, rat, chicken, fish and Drosophila. The human LOXfamily has a highly conserved C-terminal region containing the 205 aminoacid LOX catalytic domain. The conserved region contains the copperbinding (Cu), conserved cytokine receptor like domain (CRL), and thelysyl-tyrosylquinone cofactor site (LTQ). The predicted extracellularsignal sequences are represented by the hatched boxes (See FIG. 7 ofU.S. Provisional Application No. 60/963,249, which is incorporatedherein by reference). Twelve cysteine residues are also similarlyconserved, wherein two of them reside within the prepropeptide regionand ten are in the catalytically active processed form of LOX (Csiszar,Prog. Nucl. Acid Res. 70:1-32 (2001)). The conserved region alsoincludes a fibronectin binding domain.

The prepropeptide region of LOX contains the signal peptide, and iscleaved, the cleavage site predicted to be between Cys21-Ala22, togenerate a signal sequence peptide and a 48 kDa amino acid propeptideform of LOX, which is still inactive. The propeptide is N-glycosylatedduring passage through the Golgi that is secreted into the extracellularenvironment where the proenzyme, or propeptide, is cleaved betweenGly168-Asp169 by a metalloendoprotease, a procollagen C-proteinase,which are products of the Bmp1, Tll1 and Tll2 genes. BMP I (bonemorphogenetic protein I) is a procollagen C-proteinase that processesthe propeptide to yield a functional 30 kDa enzyme and an 18 kDapropeptide. The sequence coding for the propeptide is moderately(60-70%) conserved, whereas the sequence coding for the C-terminal 30kDa region of the proenzyme in which the active site is located ishighly conserved (approximately 95%). (Kagan and Li, J. Cell. Biochem.88:660-672 (2003); Kagan et al., J. Cell Biochem. 59:329-38 (1995)). TheN-glycosyl units are also subsequently removed. LOX occurs inunprocessed and/or processed (mature) forms. The mature form of LOX istypically active although, in some embodiments, unprocessed LOX is alsoactive.

Particular examples of a LOXL enzyme or protein are described in Molnaret al., Biochim Biophys Acta. 1647:220-24 (2003); Csiszar, Prog. Nucl.Acid Res. 70:1-32 (2001); and in WO 01/83702 published on Nov. 8, 2001,all of which are herein incorporated by reference. (It is noted that inthese 3 publications, “LOXL1” was referred to as “LOXL” whereas in thepresent invention “LOXL” is used to refer to a lysyl oxidase-likeproteins in general, not just LOXL1.) These enzymes include LOXL1,encoded by mRNA deposited at GenBank/EMBL BC015090; AAH15090.1; LOXL2,encoded by mRNA deposited at GenBank/EMBL U89942; LOXL3, encoded by mRNAdeposited at GenBank/EMBL AF282619; AAK51671.1; and LOXL4, encoded bymRNA deposited at GenBank/EMBL AF338441; AAK71934.1.

Similar potential signal peptides as those described above for LOX havebeen predicted at the amino terminus of LOXL, LOXL2, LOXL3, and LOXL4.The predicted signal cleavage sites are between Gly25-Gln26 for LOXL,between Ala25-Gln26, for LOXL2, and between Gly25-Ser26 for LOXL3. Theconsensus for BMP-1 cleavage in pro-collagens and pro-LOX is betweenAla/Gly-Asp, and often followed by an acidic or charged residue. Apotential cleavage site to generate active LOXL is Gly303-Asp304,however, it is then followed by an atypical Pro. LOXL3 also has apotential cleavage site at Gly447-Asp448, which is followed by an Asp,processing at this site may yield an active peptide of similar size toactive LOX. A potential cleavage site of BMP-1 was also identifiedwithin LOXL4, at residues Ala569-Asp570 (Kim et al., J. Biol. Chem.278:52071-52074 (2003)). LOXL2 may also be proteolytically cleavedanalogously to the other members of the LOXL family and secreted (Akiriet al., Cancer Res. 63:1657-1666 (2003)).

LOX and LOXL enzymes act via a ping-pong mechanism which can bedescribed by Michaelis-Menten kinetics (see FIG. 1).

An example of LOX or LOXL protein include the enzyme having an aminoacid sequence substantially identical to a polypeptide expressed ortranslated from one of the following sequences: EMBL/GenBank accessions:M94054; AAA59525.1-mRNA; 545875; AAB23549.1-mRNA; 578694;AAB21243.1-mRNA; AF039291; AAD02130.1-mRNA; BC074820; AAH74820.1-mRNA;BC074872; AAH74872.1-mRNA; M84150; AAA59541.1-Genomic DNA.

The terms “LOX” and “LOXL” also encompass functional fragments orderivatives that substantially retain enzymatic activity catalyzing thedeamination of lysyl residues. Typically, a functional fragment orderivative retains at least 50% of 60%, 70%, 80%, 90%, 95%, 99% or 100%of its lysyl oxidation activity. It is also intended that a LOX or aLOXL2 protein can include conservative amino acid substitutions that donot substantially alter its activity. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity. See, e.g.,Watson, et al., Molecular Biology of the Gene, 4th Edition, 1987, TheBenjamin/Cummings Pub. Co., p. 224. Conservative and non-conservativeamino acid substitutions have been described above.

A feature not known to be common amongst the LOX and LOXL proteins isthe scavenger receptor cysteine rich (SRCR) domains. LOX and LOXL lackSRCR domains, whereas LOXL2, LOXL3, and LOXL4 each have four SRCRdomains at the N-terminus. SRCR domains are found in secreted,transmembrane, or extracellular matrix proteins. SRCR domains are alsoknown to mediate ligand binding in a number of secreted and receptorproteins (Hoheneste et al., Nat. Struct. Biol. 6:228-232 (1999); Sasakiet al., EMBO J. 17:1606-1613 (1998)). Another domain unique to LOXL isthe presence of a proline rich domain (Molnar et al., BiochimicaBiophsyica Acta 1647:220-224 (2003)).

Tissue distribution may also differ amongst LOX and the various LOXL.LOX is highly expressed in the heart, placenta, testis, lung, kidney anduterus, but marginally in the brain and liver. LOXL1 is expressed in theplacenta, kidney, muscle, heart, lung, and pancreas, and as with LOX,has much lower expressing in the brain and liver (Kim et al., J. Biol.Chem. 270:7176-7182 (1995)). LOXL2 is highly expressed in the uterus,placenta, and other organs, but similar to LOX and LOXL, lowly expressedin the brain and liver (Jourdan Le-Saux et al., J. Biol. Chem.274:12939:12944 (1999)). LOXL3 is highly expressed in the testis,spleen, and prostate, moderately in placenta, and not in the liver,whereas LOXL4 is highly expressed in the liver (Huang et al., MatrixBiol. 20:153-157 (2001); Maki and Kivirikko, Biochem. J. 355:381-387(2001); Jourdan Le-Saux et al., Genomics 74:211-218 (2001); Asuncion etal., Matrix Biol. 20:487-491 (2001)).

The expression, or implication of LOX and the different LOXL proteins,in diseases may also vary. This may be due to a number of reasons, suchas the difference in tissue distribution, processing, domains,regulation of activity, as well as other differences between theproteins. For example, LOX and LOXL are implicated in fibrotic diseasesas both LOX and LOXL are highly expressed in myo-fibroblasts aroundfibrotic areas (Kagen, Pathol. Res. Pract. 190:910-919 (1994); Murawakiet al., Hepatology 14:1167-1173 (1991); Siegel et al., Proc. Natl. Acad.Sci. USA 75:2945-2949 (1978); Jourdan Le-Saux et al., Biochem. Biophys.Res. Comm 199:587-592 (1994); Kim et al., J. Cell Biochem. 72:181-188(1999)). LOX and the various LOXL are also implicated in a number ofcancers. For example, LOXL and LOXL4 have been shown to beepigenetically silenced and can inhibit ras/extracellularsignal-regulated kinase signaling pathway in human bladder cancer (Wu etal., Cancer Res. 67:4123-4129 (2007)). Others have shown selectiveupregulation and amplification of the LOXL4 gene in head and necksquamous cell carcinoma (Gorough et al., J. Pathol. 212:74-82 (2007)).LOX and LOXL2 have also been implicated in a number of tumors, such ascolon and esophageal cancers (Csiszar, Frog. Nucl. Acid Res. 70:1-32(2001)). In breast cancer, LOX and the LOXL family members have beenlinked to the cancer (Kirschmann et al., Cancer Res. 62:448-4483(2002)).

III. EPITHELIAL—MESENCHYMAL TRANSITION

Epithelial-to-Mesenchymal Transition (EMT) refers to the process wherebya cell with a gene expression/phenotype characteristic of epithelialcell (i.e., expressing specific proteins, factors, and molecules)changes or alters the genes or their level of expression which resultsin a change in the phenotype of the cell as exhibited by the alterationor change in the genes expressed.

Epithelial and mesenchymal cells represent distinct lineages, each witha unique gene expression profile that imparts attributes specific toeach cell type. Conversion of an epithelial cell into a mesenchymal cellrequires alterations in morphology, cellular architecture, adhesion,and/or migration capacity. Advanced tumor cells frequently exhibit aconspicuous down-regulation of epithelial markers and a loss ofintercellular junctions, resulting in a loss of epithelial polarity andreduced intercellular adhesion. The loss of epithelial features is oftenaccompanied by increased cell motility and expression of mesenchymalgenes. EMT can include loss of contact inhibition, altered growthcontrol, and/or enhanced invasiveness (Christiansen and Rajasekaran,Cancer Res., 66(17): 8319-8326 (2006); and Thiery et al., Curr. Opin.Cell. Biol., 15: 740-6 (2003)). Molecular and morphologic featuresindicative of EMT correlate with poor histologic differentiation,destruction of tissue integrity, and metastasis. EMT provides mechanismsfor epithelial cells to overcome the physical constraints imposed onthem by intercellular junctions and adopt a motile phenotype (Burdsal etal. Development, 118:829-44 (1993); and Nieto et al., Mech, Dev.,105:27-35 (2001)).

Commonly used molecular markers for EMT include increased expression ofN-cadherin and vimentin, nuclear localization of β-catenin, andincreased production of the transcription factors such as Snail1(Snail), Snail2 (Slug), Twist, EF1/ZEB1, SIP1/ZEB2, and/or E47 thatinhibit E-cadherin production. Phenotypic markers for an EMT include,but are not limited to, an increased capacity for migration andthree-dimensional invasion, as well as resistance to apoptosis. Thesemarkers have further been correlated with induction of EMT and anassociation with cancerous phenotypes.

The occurrence of EMT during tumor progression allows tumor cells toacquire the capacity to infiltrate surrounding tissue and ultimately tometastasize to distant sites. Changes in gene expression within tumorcells can indicate a progression from epithelial or epithelial-like geneexpression pattern to a mesenchymal or mesenchymal-like gene expressionpattern. By way of example, the identification of loss of E-cadherin iscorrelated with metastatic carcinoma as well as resistance to cancertherapies such as EGFR inhibitors and IGF-R1 inhibitors. Analysis ofmany different types of cancer reveals that circulating tumor cells, orthose found as micrometastases, evidence mesenchymal conversion based onchanges of expression in a set of markers. These markers include, butare not limited to, EGFR, E-cadherin, ErbB3, RAB25, integrin beta 6,cadherin-2, fibroblast growth factor binding protein 1, distal-lesshomeo box 1, ZEB1 (transcription factor 8), SIP1, and vimentin.

By way of example, an epithelial-like gene expression profile includesexpression, or increase expression of genes such as E-cadherin, ErbB3,or EGFR. An epithelial-like gene expression profile can include theexpression of one or more of these genes, at least two, or at leastthree of these genes.

As with the previously described therapy-resistant cancers, theexpression levels of E-cadherin, ErbB3, RAB25, integrin beta 6,cadherin-2, fibroblast growth factor binding protein 1, distal-lesshomeo box 1, ZEB1 (transcription factor 8), SIP1, TGF-β, FOXC2, GSK-3β,Smad-3, Pez, Snail1, Snail2, and ILK, and vimentin represent genes thatare common to EMT characteristics as well as with those epithelial-basedtumor cells/cancers that develop resistance to their respectivetherapies. The present invention also generally relates to a method totreat a patient with cancer, and particularly a cancer that hasexperienced EMT. The inventors have discovered that cancers that haveexperienced EMT or have switched from an epithelial-like gene expressionpattern to a mesenchymal-like gene expression pattern are responsive toLOX/LOXL inhibitors.

For assessment of tumor cell epithelial or mesenchymal biomarkerexpression, patient samples containing tumor cells, or proteins ornucleic acids produced by these tumor cells, can be used in methodsdescribed, for example, in U.S. patent application Publication Number20070065858, which is incorporated in its entirety by reference herein.Briefly, the level of expression of the biomarker can be assessed byassessing the amount (e.g., absolute amount or concentration) of themarker in a tumor cell sample, e.g., a tumor biopsy obtained from apatient, or other patient sample containing material derived from thetumor (e.g., blood, serum, urine, or other bodily fluids or excretionsas described herein above). The cell sample can, of course, be subjectedto a variety of well-known post-collection preparative and storagetechniques (e.g., nucleic acid and/or protein extraction, fixation,storage, freezing, ultrafiltration, concentration, evaporation,centrifugation, etc.) prior to assessing the amount of the marker in thesample. Likewise, tumor biopsies can also be subjected topost-collection preparative and storage techniques, e.g., fixation.

One can detect expression of biomarker proteins having at least oneportion which is displayed on the surface of tumor cells which expressit. It is a simple matter for the skilled artisan to determine whether amarker protein, or a portion thereof, is exposed on the cell surface.For example, immunological methods can be used to detect such proteinson whole cells, or well known computer-based sequence analysis methodscan be used to predict the presence of at least one extracellular domain(i.e., including both secreted proteins and proteins having at least onecell-surface domain). Expression of a marker protein having at least oneportion which is displayed on the surface of a cell which expresses itcan be detected without necessarily lysing the tumor cell (e.g., using alabeled antibody which binds specifically with a cell-surface domain ofthe protein).

Expression of biomarkers can be assessed by any of a wide variety ofwell known methods for detecting expression of a transcribed nucleicacid or protein. Non-limiting examples of such methods include, forexample, immunological methods for detection of secreted, cell-surface,cytoplasmic, or nuclear proteins, protein purification methods, proteinfunction or activity assays, nucleic acid hybridization methods, nucleicacid reverse transcription methods, and nucleic acid amplificationmethods.

Expression of a biomarker can be assessed using an antibody (e.g., aradio-labeled, chromophore-labeled, fluorophore-labeled, orenzyme-labeled antibody), an antibody derivative (e.g., an antibodyconjugated with a substrate or with the protein or ligand of aprotein-ligand pair (e.g., biotin-streptavidin), or an antibody fragment(e.g., a single-chain antibody, an isolated antibody hypervariabledomain, etc.) which binds specifically with a biomarker protein orfragment thereof, including a biomarker protein which has undergoneeither all or a portion of post-translational modifications to which itis normally subjected in the tumor cell (e.g., glycosylation,phosphorylation, methylation etc.).

Expression of a biomarker can also be assessed by preparing mRNA/cDNA(i.e., a transcribed polynucleotide) from cells in a patient sample, andby hybridizing the mRNA/cDNA with a reference polynucleotide which is acomplement of a biomarker nucleic acid, or a fragment thereof. cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction methods prior to hybridization with the referencepolynucleotide. Expression of one or more biomarkers can likewise bedetected using quantitative PCR to assess the level of expression of thebiomarker(s). Alternatively, any of the many known methods of detectingmutations or variants (e.g., single nucleotide polymorphisms, deletions,etc.) of a biomarker can be used to detect occurrence of a biomarker ina patient.

A mixture of transcribed polynucleotides obtained from the sample can becontacted with a substrate having fixed thereto a polynucleotidecomplementary to or homologous with at least a portion (e.g., at least7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) ofa biomarker nucleic acid. If polynucleotides complementary to, orhomologous with, are differentially detectable on the substrate (e.g.,detectable using different chromophores or fluorophores, or fixed todifferent selected positions), then the levels of expression of aplurality of biomarkers can be assessed simultaneously using a singlesubstrate (e.g., a “gene chip” microarray of polynucleotides fixed atselected positions). When a method of assessing biomarker expression isused which involves hybridization of one nucleic acid with another,hybridization can be performed under stringent hybridization conditions.

When a plurality of biomarkers of the invention are used in the methodsof the invention, the level of expression of each biomarker in a patientsample can be compared with the normal level of expression of each ofthe plurality of biomarkers in non-cancerous samples of the same type,either in a single reaction mixture (i.e., using reagents, such asdifferent fluorescent probes, for each biomarker) or in individualreaction mixtures corresponding to one or more of the biomarkers.

The level of expression of a biomarker in normal (i.e., non-cancerous)human tissue can be assessed in a variety of ways. This normal level ofexpression can be assessed by assessing the level of expression of thebiomarker in a portion of cells which appears to be non-cancerous, andthen comparing the normal level of expression with the level ofexpression in a portion of the tumor cells. As further informationbecomes available as a result of routine performance of the methodsdescribed herein, population-average values for normal expression of thebiomarkers can be used. Alternatively, the normal level of expression ofa biomarker can be determined by assessing expression of the biomarkerin a patient sample obtained from a non-cancer-afflicted patient, from apatient sample obtained from a patient before the suspected onset ofcancer in the patient, from archived patient samples, and the like.

An exemplary method for detecting the presence or absence of a biomarkerprotein or nucleic acid in a biological sample involves obtaining abiological sample (e.g., a tumor-associated body fluid) from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting the polypeptide or nucleic acid (e.g., mRNA,genomic DNA, or cDNA). The detection methods can, thus, be used todetect mRNA, protein, cDNA, or genomic DNA, for example, in a biologicalsample in vitro as well as in vivo. In vitro techniques for detection ofmRNA include, for example, Northern hybridizations and in situhybridizations. In vitro techniques for detection of a biomarker proteininclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitation and immunofluorescence. Invitro techniques for detection of genomic DNA include, for example,Southern hybridizations. In vivo techniques for detection of mRNAinclude, for example, polymerase chain reaction (PCR), Northernhybridizations and in situ hybridizations. Furthermore, in vivotechniques for detection of a biomarker protein include introducing intoa subject a labeled antibody directed against the protein or fragmentthereof. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a biomarker, anda probe, under appropriate conditions and for a time sufficient to allowthe biomarker and probe to interact and bind, thus forming a complexthat can be removed and/or detected in the reaction mixture. Theseassays can be conducted in a variety of ways.

For example, one method to conduct such an assay involves anchoring thebiomarker or probe onto a solid phase support, also referred to as asubstrate, and detecting target biomarker/probe complexes anchored onthe solid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of biomarker, can be anchored onto a carrier orsolid phase support. In another embodiment, the reverse situation ispossible, in which the probe can be anchored to a solid phase and asample from a subject can be allowed to react as an unanchored componentof the assay.

There are several established methods for anchoring assay components toa solid phase. These include, without limitation, biomarker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored. Other suitablecarriers or solid phase supports for such assays include any materialcapable of binding the class of molecule to which the biomarker or probebelongs. Well-known supports or carriers include, but are not limitedto, glass, polystyrene, nylon, polypropylene, nylon, polyethylene,dextran, amylases, natural and modified celluloses, polyacrylamides,gabbros, and magnetite. In order to conduct assays with the abovementioned approaches, the non-immobilized component is added to thesolid phase upon which the second component is anchored. After thereaction is complete, uncomplexed components can be removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized upon the solid phase. The detection of biomarker/probecomplexes anchored to the solid phase can be accomplished in a number ofmethods outlined herein. In one embodiment, the probe, when it is theunanchored assay component, can be labeled for the purpose of detectionand readout of the assay, either directly or indirectly, with detectablelabels discussed herein and which are well-known to one skilled in theart.

It is also possible to directly detect biomarker/probe complex formationwithout further manipulation or labeling of either component (biomarkeror probe), for example by utilizing the technique of fluorescence energytransfer (i.e., FET, see for example, Lakowicz et al., U.S. Pat. No.5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first, donor molecule is selected such that,upon excitation with incident light of appropriate wavelength, itsemitted fluorescent energy will be absorbed by a fluorescent label on asecond acceptor molecule, which in turn is able to fluoresce due to theabsorbed energy. Alternately, the donor protein molecule can simplyutilize the natural fluorescent energy of tryptophan residues. Labelsare chosen that emit different wavelengths of light, such that theacceptor molecule label can be differentiated from that of the donor.Since the efficiency of energy transfer between the labels is related tothe distance separating the molecules, spatial relationships between themolecules can be assessed. In a situation in which binding occursbetween the molecules, the fluorescent emission of the acceptor moleculelabel in the assay should be maximal. An FET binding event can beconveniently measured through standard fluorometric detection means wellknown in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a biomarker can be accomplished without labeling either assaycomponent (probe or biomarker) by utilizing a technology such asreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander,S. and Urbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al.,1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or“surface plasmon resonance” is a technology for studying biospecificinteractions in real time, without labeling any of the interactants(e.g., BIAcore). Changes in the mass at the binding surface (indicativeof a binding event) result in alterations of the refractive index oflight near the surface (the optical phenomenon of surface plasmonresonance (SPR)), resulting in a detectable signal which can be used asan indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with biomarker and probe as solutesin a liquid phase. In such an assay, the complexed biomarker and probeare separated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, biomarker/probe complexes can be separated fromuncomplexed assay components through a series of centrifugal steps, dueto the different sedimentation equilibria of complexes based on theirdifferent sizes and densities (see, for example, Rivas, G., and Minton,A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques can also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format; for example, the relativelylarger complex can be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thebiomarker/probe complex as compared to the uncomplexed components can beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J. Chromatogr B Biomed SciAppl Oct. 10, 1997; 699(1-2):499-525). Gel electrophoresis can also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically used.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

In another embodiment, the level of biomarker mRNA can be determinedboth by in situ and by in vitro formats in a biological sample usingmethods known in the art. The term “biological sample” is intended toinclude tissues, cells, biological fluids and isolates thereof, isolatedfrom a subject, as well as tissues, cells and fluids present within asubject. Many expression detection methods use isolated RNA. For invitro methods, any RNA isolation technique that does not select againstthe isolation of mRNA can be utilized for the purification of RNA fromtumor cells (see, e.g., Ausubel et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, New York 1987-1999). Additionally,large numbers of tissue samples can readily be processed usingtechniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One diagnosticmethod for the detection of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to the mRNAencoded by the gene being detected. The nucleic acid probe can be, forexample, a full-length cDNA, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to a mRNA or genomic DNA encoding a biomarker of the presentinvention. Other suitable probes for use in the diagnostic assays of theinvention are described herein. Hybridization of an mRNA with the probeindicates that the biomarker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the biomarkers of the present invention.

An alternative method for determining the level of mRNA biomarker in asample involves the process of nucleic acid amplification, e.g., byRT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat.No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad.Sci. USA, 88:189-193), self sustained sequence replication (Guatelli etal., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the tumorcells prior to detection. In such methods, a cell or tissue sample isprepared and/or processed using known histological methods. The sampleis then immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes thebiomarker.

As an alternative to making determinations based on the absoluteexpression level of the biomarker, determinations can be based on thenormalized expression level of the biomarker. Expression levels arenormalized by correcting the absolute expression level of a biomarker bycomparing its expression to the expression of a gene that is not abiomarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a patientsample, to another sample, e.g., a non-tumor sample, or between samplesfrom different sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of abiomarker (e.g., a mesenchymal biomarker), the level of expression ofthe biomarker is determined for 10 or more, 20 or more, 30 or more, 40or more, or 50 or more samples of normal versus cancer cell isolatesprior to the determination of the expression level for the sample inquestion. The mean expression level of each of the genes assayed in thelarger number of samples is determined and this is used as a baselineexpression level for the biomarker. The expression level of thebiomarker determined for the test sample (absolute level of expression)is then divided by the mean expression value obtained for thatbiomarker. This provides a relative expression level.

In another embodiment of the present invention, a biomarker protein isdetected. One type of agent for detecting biomarker protein of theinvention is an antibody capable of binding to such a protein or afragment thereof such as, for example, a detectably labeled antibody.Antibodies can be polyclonal or monoclonal. An intact antibody, or anantigen binding fragment thereof (e.g., Fab, F(ab′)2, Fv, scFv, singlebinding chain polypeptide) can be used. The term “labeled,” with regardto the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) adetectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

Proteins from tumor cells can be isolated using techniques that are wellknown to those of skill in the art. The protein isolation methodsemployed can, for example, be such as those described in Harlow and Lane(Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

A variety of formats can be employed to determine whether a samplecontains a protein that binds to a given antibody. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (ELISA). A skilled artisan can readily adapt knownprotein/antibody detection methods for use in determining whether tumorcells express a biomarker of the present invention.

In one format, antibodies, or antibody fragments or derivatives, can beused in methods such as Western blots or immunofluorescence techniquesto detect the expressed proteins. In such uses, either the antibody orproteins can be immobilized on a solid support. Suitable solid phasesupports or carriers include any support capable of binding an antigenor an antibody. Well-known supports or carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. One skilled in the art will know many other suitable carriersfor binding antibody or antigen, and will be able to adapt such supportfor use with the present invention. For example, protein isolated fromtumor cells can be run on a polyacrylamide gel electrophoresis andimmobilized onto a solid phase support such as nitrocellulose. Thesupport can then be washed with suitable buffers followed by treatmentwith the detectably labeled antibody. The solid phase support can thenbe washed with the buffer a second time to remove unbound antibody. Theamount of bound label on the solid support can then be detected byconventional means.

For ELISA assays, specific binding pairs can be of the immune ornon-immune type. Immune specific binding pairs are exemplified byantigen-antibody systems or hapten/anti-hapten systems. There can bementioned fluorescein/anti-fluorescein,dinitrophenyl/anti-dinitrophenyl, biotin/anti-biotin,peptide/anti-peptide and the like. The antibody member of the specificbinding pair can be produced by customary methods familiar to thoseskilled in the art. Such methods involve immunizing an animal with theantigen member of the specific binding pair. If the antigen member ofthe specific binding pair is not immunogenic, e.g., a hapten, it can becovalently coupled to a carrier protein to render it immunogenic.Non-immune binding pairs include systems wherein the two componentsshare a natural affinity for each other but are not antibodies.Exemplary non-immune pairs are biotin-streptavidin, intrinsicfactor-vitamin B12, folic acid-folate binding protein and the like.

A variety of methods are available to covalently label antibodies withmembers of specific binding pairs. Methods are selected based upon thenature of the member of the specific binding pair, the type of linkagedesired, and the tolerance of the antibody to various conjugationchemistries. Biotin can be covalently coupled to antibodies by utilizingcommercially available active derivatives. Some of these arebiotin-N-hydroxy-succinimide which binds to amine groups on proteins;biotin hydrazide which binds to carbohydrate moieties, aldehydes andcarboxyl groups via a carbodiimide coupling; and biotin maleimide andiodoacetyl biotin which bind to sulfhydryl groups. Fluorescein can becoupled to protein amine groups using fluorescein isothiocyanate.Dinitrophenyl groups can be coupled to protein amine groups using2,4-dinitrobenzene sulfate or 2,4-dinitrofluorobenzene. Other standardmethods of conjugation can be employed to couple monoclonal antibodiesto a member of a specific binding pair including dialdehyde,carbodiimide coupling, homofunctional cross-linking, andheterobifunctional cross-linking. Carbodiimide coupling is an effectivemethod of coupling carboxyl groups on one substance to amine groups onanother. Carbodiimide coupling is facilitated by using the commerciallyavailable reagent 1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDAC).

Homobifunctional cross-linkers, including the bifunctional imidoestersand bifunctional N-hydroxysuccinimide esters, are commercially availableand are employed for coupling amine groups on one substance to aminegroups on another. Heterobifunctional cross-linkers are reagents whichpossess different functional groups. The most common commerciallyavailable heterobifunctional cross-linkers have an amine reactiveN-hydroxysuccinimide ester as one functional group, and a sulfhydrylreactive group as the second functional group. The most commonsulfhydryl reactive groups are maleimides, pyridyl disulfides and activehalogens. One of the functional groups can be a photoactive arylnitrene, which upon irradiation reacts with a variety of groups.

The detectably-labeled antibody or detectably-labeled member of thespecific binding pair is prepared by coupling to a reporter, which canbe a radioactive isotope, enzyme, fluorogenic, chemiluminescent orelectrochemical materials. Two commonly used radioactive isotopes are125I and 3H. Standard radioactive isotopic labeling procedures includethe chloramine T, lactoperoxidase and Bolton-Hunter methods for 125I andreductive methylation for 3H. The term “detectably-labeled” refers to amolecule labeled in such a way that it can be readily detected by theintrinsic enzymatic activity of the label or by the binding to the labelof another component, which can itself be readily detected.

Enzymes suitable for use in this invention include, but are not limitedto, horseradish peroxidase, alkaline phosphatase, β-galactosidase,glucose oxidase, luciferases, including firefly and renilla,β-lactamase, urease, green fluorescent protein (GFP) and lysozyme.Enzyme labeling is facilitated by using dialdehyde, carbodiimidecoupling, homobifunctional cross-linkers and heterobifunctionalcross-linkers as described above for coupling an antibody with a memberof a specific binding pair.

The labeling method chosen depends on the functional groups available onthe enzyme and the material to be labeled, and the tolerance of both tothe conjugation conditions. The labeling method used in the presentinvention can be one of, but not limited to, any conventional methodscurrently employed including those described by Engvall and Pearlmann,Immunochemistry 8, 871 (1971), Avrameas and Temynck, Immunochemistry 8,1175 (1975), Ishikawa et al., J. Immunoassay 4(3):209-327 (1983) andJablonski, Anal. Biochem. 148:199 (1985).

Labeling can be accomplished by indirect methods such as using spacersor other members of specific binding pairs. An example of this is thedetection of a biotinylated antibody with unlabeled streptavidin andbiotinylated enzyme, with streptavidin and biotinylated enzyme beingadded either sequentially or simultaneously. Thus, according to thepresent invention, the antibody used to detect can be detectably-labeleddirectly with a reporter or indirectly with a first member of a specificbinding pair. When the antibody is coupled to a first member of aspecific binding pair, then detection is effected by reacting theantibody-first member of a specific binding complex with the secondmember of the binding pair that is labeled or unlabeled as mentionedabove.

Moreover, the unlabeled detector antibody can be detected by reactingthe unlabeled antibody with a labeled antibody specific for theunlabeled antibody. In this instance “detectably-labeled” as used aboveis taken to mean containing an epitope by which an antibody specific forthe unlabeled antibody can bind. Such an anti-antibody can be labeleddirectly or indirectly using any of the approaches discussed above. Forexample, the anti-antibody can be coupled to biotin which is detected byreacting with the streptavidin-horseradish peroxidase system discussedabove. Thus, in one embodiment, biotin is utilized. The biotinylatedantibody is in turn reacted with streptavidin-horseradish peroxidasecomplex. Orthophenylenediamine, 4-chloro-naphthol, tetramethylbenzidine(TMB), ABTS, BTS or ASA can be used for chromogenic detection.

In one immunoassay format for practicing this invention, a forwardsandwich assay is used in which the capture reagent has beenimmobilized, using conventional techniques, on the surface of a support.Suitable supports used in assays include synthetic polymer supports,such as polypropylene, polystyrene, substituted polystyrene, e.g.,aminated or carboxylated polystyrene, polyacrylamides, polyamides,polyvinylchloride, glass beads, agarose, or nitrocellulose.

IV. ANTI-LOX ANTIBODIES AND ANTI-LOXL2 ANTIBODIES

Provided herein are antibodies that can be used to diagnose angiogenesisand associated diseases, fibrosis and associated diseases, tumors ormetastasis. Provided herein are antibodies that inhibit angiogenesis andassociated diseases, inhibit fibrosis and associated diseases, and treattumors or metastasis. Provided herein are antibodies that can be used tomonitor efficacy of treatment regimens and protocols and the like asdescribed throughout the present application and known in the art.Antibodies and antigen binding fragments useful in such methods arethose, for example, that specifically bind LOX or LOXL2.

The disclosure also describes cell lines which produce the antibodies orfunctional fragments thereof, methods for producing the cell lines, andmethods for producing the antibodies or functional fragments thereof.

The term “antibody” or “antibody molecule” in the various grammaticalforms is used herein as a collective noun that refers to a population ofimmunoglobulin molecules and/or immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope. Thus, reference to an “antibody” alsoincludes reference to any of the antigen binding fragments ofantibodies.

As used herein, “immunoreactive” refers to antibodies or fragmentsthereof that are specific to a sequence of amino acid residues (“bindingsite” or “epitope”), yet if are cross-reactive to otherpeptides/proteins, are not toxic at the levels at which they areformulated for administration to human use. “Epitope” refers to thatportion of an antigen capable of forming a binding interaction with anantibody or antigen binding fragment thereof. Such binding interactioncan be manifested as an intermolecular contact with one or more aminoacid residues of a CDR. Antigen binding can involve a CDR3 or a CDR3pair. An epitope can be a linear peptide sequence (i.e., “continuous”)or can be composed of noncontiguous amino acid sequences (i.e.,“conformational” or “discontinuous”). The term “preferentially binds”means that the binding agent binds to the binding site with greateraffinity than it binds unrelated amino acid sequences.

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as adissociation constant (Kd). Affinity can be at least 1-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, at least10-fold greater, at least 20-fold greater, at least 30-fold greater, atleast 40-fold greater, at least 50-fold greater, at least 60-foldgreater, at least 70-fold greater, at least 80-fold greater, at least90-fold greater, at least 100-fold greater, or at least 1000-foldgreater, or more, than the affinity of an antibody for unrelated aminoacid sequences. Affinity of an antibody to a target protein can be, forexample, from about 100 nanomolar (nM) to about 0.1 nM, from about 100nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar(fM) or more. As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. The terms “immunoreactive” and “preferentially binds” are usedinterchangeably herein with respect to antibodies and/or antigen-bindingfragments.

The term “antibody” also includes molecules which have been engineeredthrough the use of molecular biological technique to include onlyportions of the native molecule as long as those molecules have theability to bind a particular antigen or sequence of amino acids with therequired specificity. Such alternative antibody molecules includeclassically known portions of the antibody molecules, single chainantibodies, and single chain binding molecules.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.These particular regions have been described by Kabat et al., J. Biol.Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and HumanServices, “Sequences of proteins of immunological interest” (1991); byChothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or grafted antibodies or variants thereof is intended tobe within the scope of the term as defined and used herein. The aminoacid residues which encompass the CDRs as defined by each of the abovecited references are set forth below in Table 1 as a comparison.

TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally a discontinuous amino acidsequence between about 100-120 amino acids in length but is intended toreference only those amino acids outside of the CDRs. As used herein,the term “framework region” is intended to mean each domain of theframework that is separated by the CDRs.

“An inhibitor of LOX activity” or “an inhibitor of LOXL2 activity” canbe an antibody or an antigen binding fragment thereof that directly orindirectly inhibits activity of lysyl oxidase, including but not limitedto gene expression, post-translation modification, enzymatic processingor cleavage, binding to a modulator of LOX/LOXL2, enzymatic activity ofLOX/LOXL2 or any other activity described herein.

As used herein, the term “antibody” refers to an isolated or recombinantbinding agent that comprises the necessary variable region sequences tospecifically bind an antigenic epitope. Therefore, an antibody is anyform of antibody or fragment thereof that exhibits the desiredbiological activity, e.g., binding the specific target antigen. Thus, itis used in the broadest sense and specifically covers monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, human antibodies, humanized antibodies, chimeric antibodies,diabodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments including but not limited to single chain bindingpolypeptides, VH, VL, Fv, scFv, Fab, and Fab2, etc., so long as theyexhibit the desired biological activity. The term “human antibody”therefore refers to antibodies containing sequences of human origin,except for possible non-human CDR regions, and does not imply that thefull structure of an Ig molecule be present, only that the antibody hasminimal immunogenicity in a human.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. The term “specific binding” is applicable toa situation in which an antibody or antigen binding fragment thereofdoes not show any significant binding to molecules other than itsepitope. In one embodiment, an antibody or antigen binding fragmentthereof specifically binds to a human LOX or to human LOXL2 with adissociation constant Kd equal to or lower than about 100 nM, lower thanabout 10 nM, lower than about 1 nM, lower than about 0.5 nM, lower thanabout 0.1 nM, lower than about 0.01 nM, or lower than about 0.005 nMmeasured at a temperature of about 4° C., 25° C., 37° C. or 42° C.

Conventional methods can be used to prepare antibodies. For example, byusing a peptide or full length lysyl oxidase protein, polyclonalantisera or monoclonal antibodies can be made using standard methods. Amammal, (e.g., a mouse, hamster, or rabbit) can be immunized with animmunogenic form of the peptide that elicits an antibody response in themammal. Techniques for conferring enhanced immunogenicity on a peptideinclude conjugation to carriers or other techniques well known in theart. For example, the protein or peptide can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassay procedures can be used with the immunogen as antigen toassess the levels of antibodies. Following immunization, antisera can beobtained and, if desired, polyclonal antibodies isolated from the sera.

The antibodies can be generated in cell culture, in phage, or in variousanimals, including but not limited to cows, rabbits, goats, mice, rats,hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes.Therefore, an antibody useful in the present methods is typically amammalian antibody. Phage techniques can be used to isolate an initialantibody or to generate variants with altered specificity or aviditycharacteristics. Such techniques are routine and well known in the art.In one embodiment, the antibody is produced by recombinant means knownin the art. For example, a recombinant antibody can be produced bytransfecting a host cell with a vector comprising a DNA sequenceencoding the antibody. One or more vectors can be used to transfect theDNA sequence expressing at least one VL and one VH region in the hostcell. Exemplary descriptions of recombinant means of antibody generationand production include Delves, Antibody Production: Essential Techniques(Wiley, 1997); Shephard, et al., Monoclonal Antibodies (OxfordUniversity Press, 2000); and Goding, Monoclonal Antibodies: Principlesand Practice (Academic Press, 1993).

Antibodies also can be made according to the protocol described by inKenney, et al. (“Production of monoclonal antibodies using a secretioncapture report web.” Biotechnology 13:787-790, 1995). Briefly, mice areinjected subcutaneously (s.c.), with antigen in an adjuvant formulation.For peptide antigens, peptides are conjugated to bovine serum albuminand formulated in Freund's Adjuvant (FA) prior to immunization. Forprotein antigens, the protein is formulated in Alhydrogel-MuramylDipeptide (ALD/MDP) adjuvant. Cells from the spleen and lymph nodes ofthe mice are isolated and fused with appropriate cells and cultured. Ahybridoma library of HAT-selected cells is isolated and is cloned. Cellsare sorted and sera and supernatants are screened for the presence ofantibodies.

“Antibody fragments” comprise a portion of an intact antibody, and caninclude the antigen binding or variable region of an intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fv fragments,scFv fragments, diabodies, linear antibodies (Zapata et al., ProteinEng. 8(10): 1057-1062 (1995)), single-chain antibody molecules, singlechain binding polypeptides, and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, each with asingle antigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)2 fragment that has two antigen combining sites and is stillcapable of cross-linking antigen.

A “single chain binding polypeptide” refers to a polypeptide having aheavy chain variable region, a light chain variable region and,optionally, an immunoglobulin Fc region. Such a molecule is a singlechain variable fragment optionally having effector function through thepresence of the immunoglobulin Fc region. Methods of preparing singlechain binding polypeptides are known in the art (e.g., US. PatentApplication 2005/0238646).

“Fv” refers to an antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

A “Fab” fragment contains a “Fv” and also contains the constant domainof the light chain and the first constant domain (CH1) of the heavychain. Fab fragments differ from Fab′ fragments by the addition of a fewresidues at the carboxy terminus of the heavy chain CH1 domain includingone or more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)2 antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains (Fc) that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa or (“κ”) and lambda or (“λ”), based on the amino acid sequences oftheir constant domains.

“Single-chain Fv” or “sFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. A Fv polypeptide can further include, if needed, apolypeptide linker between the VH and VL domains, which enables the sFvto form a desired structure for antigen binding. For a review of sFv,see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments contain a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

An antibody can also be a bispecific antibody. Bispecific antibodies aremonoclonal, chimeric, human or humanized antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is, for example, LOX or LOXL2, theother one is for any other antigen, such as, for example, a cell-surfaceprotein or receptor or receptor subunit. In additional embodiments, abispecific antibody is specific for LOX and LOXL2.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immununoglobulinheavy-chain/light-chain pairs, where the two heavy chains have differentspecificities (Milstein and Cuello, Nature, 305:537 539 (1983)). Becauseof the random assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of ten differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule is usuallyaccomplished by affinity chromatography steps. Similar procedures aredisclosed in WO 93/08829, published 13 May 1993, and in Traunecker etal., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences using conventional methods known in the art.The fusion is, generally, with an immunoglobulin heavy-chain constantdomain, containing at least part of the hinge, CH2, and CH3 regions. Inone embodiment, the first heavy-chain constant region (CH1) containingthe site necessary for light-chain binding is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy-chain fusions and,if desired, the immunoglobulin light chain, can be inserted intoseparate expression vectors, and can be co-transfected into a suitablehost organism. For further details of generating bispecific antibodiessee, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers that are recovered from recombinant cellculture. The interface can comprise at least a part of the CH3 region ofan antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g., F(ab′)2 bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)2 fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells over-expressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker that is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven LOX or LOXL2 polypeptide herein. Alternatively, an anti-LOX oranti-LOXL2 polypeptide arm may be combined with an arm which binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such asFcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellulardefense mechanisms to the cell expressing a particular targetpolypeptide. Bispecific antibodies can also be used to localizecytotoxic agents to cells that express a particular target polypeptide.These antibodies possess a target-binding arm and an arm that binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA,or TETA. Another bispecific antibody of interest binds the targetpolypeptide and further binds tissue factor (TF).

The anti-LOX antibody or anti-LOXL2 antibody can also be aheteroconjugate antibody. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980) and for treatment of HIV infection (WO 91/00360 and WO92/200373). Antibodies can be prepared in vitro using known methods insynthetic protein chemistry, including those involving cross-linkingagents. For example, immunotoxins can be constructed using a disulfideexchange reaction or by forming a thioether bond. Examples of suitablereagents for this purpose include, but are not limited to, iminothiolateand methyl-4-mercaptobutyrimidate and those disclosed, for example, inU.S. Pat. No. 4,676,980.

It may be desirable to modify an anti-LOX antibody or anti-LOXL2antibody with respect to effector function, so as to enhance, e.g., theeffectiveness of the antibody in treating or preventing cancermetastasis. For example, cysteine residue(s) may be introduced into theFc region, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity can also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research, 53: 2560-2565 (1993).Alternatively, an antibody can be engineered that has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

An “isolated antibody” is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and can include, for example, enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In one embodiment, an antibody will bepurified (1) to greater than 80%, 85%, 90%, 95%, or 99% by weight ofantibody as determined by the Lowry method, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, and/or (3) to homogeneityby SDS-PAGE under reducing or non-reducing conditions using Coomassieblue or silver stain. The term “isolated antibody” includes within itsscope an antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Generally, isolation of an antibody or antigen binding fragment thereofwill include at least one purification step.

An antibody can be a humanized antibody or a human antibody. Humanizedforms of non-human (e.g., murine) antibodies include, for example,chimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, scFv, Fab, Fab′, F(ab′)2, single chain binding polypeptide,VH, VL, or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin. Chimericantibodies include those in which the heavy and light chain variableregions are combined with human constant regions (Fc). Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues that are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence.

A humanized antibody can also contain at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” or “donor”residues, which are typically taken from an “import” or “donor” variabledomain. Humanization can be essentially performed following the methodof Winter and co-workers (Jones et al., Nature, 321:522 525 (1986);Riechmann et al., Nature, 332:323 327 (1988)); Verhoeyen et al. Science,239:1534 1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies include chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)). Similarly,human antibodies can be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812 13 (1994); Fishwald et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13: 65-93 (1995).

Murine monoclonal antibodies have been developed that bind LOX or LOXL2and block the enzymatic activity thereof. Humanized antibodies andantigen-binding fragments thereof described herein are created byhumanization of the VL and VH sequences of the murine monoclonalanti-LOX and anti-LOXL2 antibodies.

Humanized immunoglobulins, including humanized antibodies, have beenconstructed by means of genetic engineering. Most humanizedimmunoglobulins that have been previously described have comprised aframework that is identical to the framework of a particular humanimmunoglobulin chain (i.e., an acceptor or recipient), and three CDRsfrom a non-human (donor) immunoglobulin chain. As described herein,humanization can also include criteria by which a limited number ofamino acids in the framework of a humanized immunoglobulin chain areidentified and chosen to be the same as the amino acids at thosepositions in the donor rather than in the acceptor, in order to increasethe affinity of an antibody comprising the humanized immunoglobulinchain.

The present invention is based in part on the model that twocontributing causes of the loss of affinity in prior means of producinghumanized antibodies (using as examples mouse antibodies as the sourceof CDRs) are: (1) when the mouse CDRs are combined with a humanframework, the amino acids in the frameworks close to the CDRs becomehuman instead of mouse. Without intending to be bound by theory, thesechanged amino acids may slightly distort the CDRs (e.g., they may createdifferent electrostatic or hydrophobic forces than in the donor mouseantibody, and the distorted-CDRs may not make as effective contacts withthe antigen as the CDRs did in the donor antibody); (2) also, aminoacids in the original mouse antibody that are close to, but not part of,the CDRs (i.e., still part of the framework), may make contacts with theantigen that contribute to affinity. These amino acids are lost when theantibody is humanized because, generally, all framework amino acids aremade human. To circumvent these issues, and to produce humanizedantibodies that have a very strong affinity for a desired antigen,humanized antibodies and antigen-binging fragments thereof can beconstructed using one or more of the following principles.

One principle is that as acceptor, a framework is used from a particularhuman immunoglobulin that is unusually homologous to the donorimmunoglobulin to be humanized, or use a consensus framework from manyhuman antibodies is used as an acceptor. For example, comparison of thesequence of a mouse heavy (or light) chain variable region against humanheavy (or light) variable regions in a data bank (for example, theNational Biomedical Research Foundation Protein Identification Resourceor the protein sequence database of the National Center forBiotechnology Information—NCBI) shows that the extent of homology todifferent human regions can vary greatly, for example from about 40% toabout 60%, about 70%, about 80%, or higher. By choosing as the acceptorimmunoglobulin one of the human heavy chain variable regions that ismost homologous to the heavy chain variable region of the donorimmunoglobulin, fewer amino acids will be changed in going from thedonor immunoglobulin to the humanized immunoglobulin. By choosing as theacceptor immunoglobulin one of the human light chain variable regionsthat is most homologous to the light chain variable region of the donorimmunoglobulin, fewer amino acids will be changed in going from thedonor immunoglobulin to the humanized immunoglobulin. Generally, usingsuch techniques, there is a reduced chance of changing an amino acidnear one or more of the CDRs that distorts their conformation. Moreover,the precise overall shape of a humanized antibody comprising thehumanized immunoglobulin chain may more closely resemble the shape ofthe donor antibody, thereby also reducing the chance of distorting theCDRS.

One can also use light and heavy chains from the same human antibody asacceptor sequences, to improve the likelihood that the humanized lightand heavy chains will make favorable contacts with each other.Alternatively, one can also use light and heavy chains from differenthuman antibody germline sequences as acceptor sequences; when suchcombinations are used, one can readily determine whether the VH and VLbind an epitope of interest using conventional assays (e.g., an ELISA).In one example, the human antibody will be chosen in which the light andheavy chain variable regions sequences, taken together, are overall mosthomologous to the donor light and heavy chain variable region sequences.Sometimes greater weight will be given to the heavy chain sequence.Regardless of how the acceptor immunoglobulin is chosen, higher affinitycan, in some cases, be achieved by selecting a small number of aminoacids in the framework of the humanized immunoglobulin chain to be thesame as the amino acids at those positions in the donor rather than inthe acceptor. Methods of affinity maturation are known in the art.

Humanized antibodies generally have at least three potential advantagesover mouse or chimeric antibodies for use in human therapy. Because theeffector portion of an antibody is human, it is believed to interactbetter with the other parts of the human immune system (e.g., destroythe target cells more efficiently by complement-dependent cytotoxicity(CDC) or antibody-dependent cellular cytotoxicity (ADCC)). Additionally,the human immune system should not recognize the framework or constantregion of the humanized antibody as foreign, and therefore the antibodyresponse against such an injected antibody should be less than against atotally foreign mouse antibody or a partially foreign chimeric antibody.Finally, mouse antibodies are known to have a half-life in the humancirculation that is much shorter than the half-life of human antibodies.Humanized antibodies can, presumably, have a half-life more similar tonaturally-occurring human antibodies, allowing smaller and less frequentdoses to be given.

Humanization of antibodies and antigen-binding fragments thereof, can beaccomplished via a variety of methods known in the art and describedherein. Similarly, production of humanized antibodies can also beaccomplished via methods known in the art and described herein.

Methods for modifications of framework regions are known in the art andare contemplated herein. Selection of one or more relevant frameworkamino acid positions to be altered depends on a variety of criteria. Onecriterion for selecting relevant framework amino acids to change can bethe relative differences in amino acid framework residues between thedonor and acceptor molecules. Selection of relevant framework positionsto alter using this approach has the advantage of avoiding anysubjective bias in residue determination or any bias in CDR bindingaffinity contribution by the residue.

Another criterion that can be used for determining the relevant aminoacid positions to change can be, for example, selection of frameworkresidues that are known to be important or to contribute to CDRconformation. For example, canonical framework residues are importantfor CDR conformation and/or structure. Targeting of a canonicalframework residue as a relevant position to change can be used toidentify a more compatible amino acid residue in context with itsassociated donor CDR sequence.

The frequency of an amino acid residue at a particular frameworkposition is another criterion which can be used for selecting relevantframework amino acid positions to change. For example, comparison of theselected framework with other framework sequences within its subfamilycan reveal residues that occur at minor frequencies at a particularposition or positions. Positions harboring less abundant residues aresimilarly applicable for selection as a position to alter in theacceptor variable region framework.

The relevant amino acid positions to change also can be selected, forexample, based on proximity to a CDR. In certain contexts, FR residuescan participate in CDR conformation and/or antigen binding. Moreover,this criterion can similarly be used to prioritize relevant positionsselected by other criteria described herein. Therefore, differentiatingbetween residues proximal and distal to one or more CDRs represents oneway to reduce the number of relevant positions to change.

Other criteria for selecting relevant amino acid framework positions toalter include, for example, residues that are known or predicted toreside in a three dimensional space near the antigen-CDR interface orpredicted to modulate CDR activity. Similarly, framework residues thatare known to, or predicted to, form contacts between the heavy (V_(H))and light (V_(L)) chain variable region interface can be selected. Suchframework positions can affect the conformation and/or affinity of a CDRby modulating the CDR binding pocket, antigen (epitope) interaction orthe V_(H) and V_(L) interaction. Therefore, selection of these aminoacid positions for constructing a diverse population for screening ofbinding activity can be used to identify framework changes which replaceresidues having detrimental effects on CDR conformation or compensatefor detrimental effects of residues occurring elsewhere in theframework.

Other framework residues that can be selected for alteration includeamino acid positions that are inaccessible to solvent. Such residues aregenerally buried in the variable region and are, therefore, capable ofinfluencing the conformation of the CDR or V_(H) and V_(L) interactions.Solvent accessibility can be predicted, for example, from the relativehydrophobicity of the environment created by the amino acid side chainsof the polypeptide and/or by known three-dimensional structural data.

Following selection of relevant amino acid positions in the donor CDRs,as well as any relevant amino acid positions in the framework regionsdesired to be varied, amino acid changes at some or all of the selectedpositions can be incorporated into encoding nucleic acids for theacceptor variable region framework and donor CDRs. Altered framework orCDR sequences can be individually made and tested, or can besequentially or simultaneously combined and tested.

The variability at any or all of the altered positions can range from afew to a plurality of different amino acid residues, including alltwenty naturally occurring amino acids or functional equivalents andanalogues thereof. In some cases, non-naturally occurring amino acidsmay also be considered and are known in the art.

Selection of the number and location of the amino acid positions to varyis flexible and can depend on the intended use and desired efficiencyfor identification of the altered variable region having a desirableactivity such as substantially the same or greater binding affinitycompared to the donor variable region. In this regard, the greater thenumber of changes that are incorporated into an altered variable regionpopulation, the more efficient it is to identify at least one speciesthat exhibits a desirable activity, for example, substantially the sameor greater binding affinity as the donor. Alternatively, where the userhas empirical or actual data to the affect that certain amino acidresidues or positions contribute disproportionally to binding affinity,then it can be desirable to produce a limited population of alteredvariable regions which focuses on changes within or around thoseidentified residues or positions.

For example, if CDR grafted variable regions are desired, a large,diverse population of altered variable regions can include all thenon-identical framework region positions between the donor and acceptorframework and all single CDR amino acid position changes. Alternatively,a population of intermediate diversity can include subsets, for example,of only the proximal non-identical framework positions to beincorporated together with all single CDR amino acid position changesto, for example, increase affinity of the humanized antibodies orantigen binding fragments. The diversity of the above populations can befurther increased by, for example, additionally including all pair-wiseCDR amino acid position changes. In contrast, populations focusing onpredetermined residues or positions which incorporate variant residuesat as few as one framework and/or one CDR amino acid position cansimilarly be constructed for screening and identification of an alteredantibody variable region. As with the above populations, the diversityof such focused populations can be further increased by additionallyexpanding the positions selected for change to include other relevantpositions in either or both of the framework and CDR regions. There arenumerous other combinations ranging from few changes to many changes ineither or both of the framework regions and CDRs that can additionallybe employed, all of which will result in a population of alteredvariable regions that can be screened for the identification of at leastone CDR grafted altered variable region having desired activity, forexample, binding activity to LOX/LOXL2. Those skilled in the art willknow, or can determine, which selected residue positions in theframework or donor CDRs, or subsets thereof, can be varied to produce apopulation for screening and identification of an altered antibody ofthe invention given the teachings and guidance provided herein. Codonsencoding amino acids are known in the art.

Humanized antibodies and antigen-binding fragments can be made usingconventional techniques known in the art. In addition, recombinantlyprepared antibodies can often be produced in large quantities,particularly when utilizing high level expression vectors.

Antibodies can be sequenced using conventional techniques known in theart and the amino acid sequences of the complementarity determiningregions (CDRs) determined. In one aspect, the amino acid sequences ofone or more of the CDRs is inserted into a synthetic sequence of, forexample, a human antibody (or antigen-binding fragment thereof)framework to create a human antibody that could limit adverse sidereactions of treating a human patient with a non-human antibody. Theamino acid sequences of one or more of the CDRs can also be insertedinto a synthetic sequence of, for example, into a binding protein suchas an Avimer™ to create a construct for administration to a humanpatient. Such techniques can be modified depending on the species ofanimal to be treated. For example, for veterinary uses, an antibody,antigen-binding fragment or binding protein can be synthesized foradministration of a primate, a cow, a horse, etc.

In another aspect, using art-recognized techniques such as thoseprovided and incorporated herein, nucleotides encoding amino acidsequences of one or more of the CDRs can inserted, for example, byrecombinant techniques in restriction endonuclease sites of an existingpolynucleotide that encodes an antibody, antigen-binding fragment orbinding protein.

For high level production, the most widely used mammalian expressionsystem is one which utilizes the gene amplification procedure offered bydihydrofolate reductase deficient (“dhfr−”) Chinese hamster ovary cells.The system is well known to the skilled artisan. The system is basedupon the dihydrofolate reductase “dhfr” gene, which encodes the DHFRenzyme, which catalyzes conversion of dihydrofolate to tetrahydrofolate.In order to achieve high production, dhfr− CHO cells are transfectedwith an expression vector containing a functional dhfr gene, togetherwith a gene that encodes a desired protein. In this case, the desiredprotein is recombinant antibody heavy chain and/or light chain.

By increasing the amount of the competitive DHFR inhibitor methotrexate(MTX), the recombinant cells develop resistance by amplifying the dhfrgene. In standard cases, the amplification unit employed is much largerthan the size of the dhfr gene, and as a result the antibody heavy chainis co-amplified.

When large scale production of the protein, such as the antibody chain,is desired, both the expression level and the stability of the cellsbeing employed are taken into account. In long term culture, recombinantCHO cell populations lose homogeneity with respect to their specificantibody productivity during amplification, even though they derive froma single, parental clone.

The present application provides an isolated polynucleotide (nucleicacid) encoding an antibody or antigen-binding fragment as describedherein, vectors containing such polynucleotides, and host cells andexpression systems for transcribing and translating such polynucleotidesinto polypeptides.

The present application also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes which compriseat least one polynucleotide as above.

The present application also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyantibody or antigen-binding fragments thereof described herein asprovided itself forms an aspect of the present application, as does amethod of production of the antibody or antigen-binding fragmentsthereof described herein which method comprises expression from encodingnucleic acid therefrom. Expression can conveniently be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression, an antibody orantigen-binding fragment can be isolated and/or purified using anysuitable technique, then used as appropriate.

Specific antibodies, antigen-binding fragments, and encoding nucleicacid molecules and vectors described herein can be provided isolatedand/or purified, e.g., from their natural environment, in substantiallypure or homogeneous form, or, in the case of nucleic acid, free orsubstantially free of nucleic acid or genes origin other than thesequence encoding a polypeptide with the required function. Nucleic acidcan comprise DNA or RNA and can be wholly or partially synthetic.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common bacterial hostis E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Plückthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of the antibodies and antigen-bindingfragments described herein, see for recent reviews, for example Raff, M.E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995)Curr. Opinion Biotech 6: 553-560, each of which is which is incorporatedherein by reference in its entirety.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors can be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference in their entirety.

Thus, a further aspect provides a host cell containing nucleic acid asdisclosed herein. A still further aspect provides a method comprisingintroducing such nucleic acid into a host cell. The introduction canemploy any available technique. For eukaryotic cells, suitabletechniques can include, for example, calcium phosphate transfection,DEAE Dextran, electroporation, biolistics, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.,vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques can include, for example, calcium chloridetransformation, electroporation and transfection using bacteriophage.

The introduction can be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid is integrated into the genome (e.g.chromosome) of the host cell. Integration can be promoted by inclusionof sequences which promote recombination with the genome, in accordancewith standard techniques.

The present application also provides a method which comprises using aconstruct as stated above in an expression system in order to expressthe antibodies or antigen-binding fragments thereof as above.

The present application also relates to isolated nucleic acids, such asrecombinant DNA molecules or cloned genes, or degenerate variantsthereof, mutants, analogs, or fragments thereof, which encode anantibody or antigen-binding sequence that binds LOX or LOXL2 describedherein.

In one aspect, the present application provides a nucleic acid whichcodes for an antibody or antigen-binding fragment thereof which bindsLOX or LOXL2 as described herein.

In a further embodiment, the full DNA sequence of the recombinant DNAmolecule or cloned gene of an antibody or antigen-binding fragmentdescribed herein can be operatively linked to an expression controlsequence which can be introduced into an appropriate host. Theapplication accordingly extends to unicellular hosts transformed withthe cloned gene or recombinant DNA molecule comprising a DNA sequenceencoding the V_(H) and/or V_(L), or portions thereof, of the antibody.

Another feature is the expression of the DNA sequences disclosed herein.As is well known in the art, DNA sequences can be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host.

Such operative linking of a DNA sequence to an expression controlsequence, of course, includes, if not already part of the DNA sequence,the provision of an initiation codon, ATG, in the correct reading frameupstream of the DNA sequence.

Polynucleotides and vectors can be provided in an isolated and/or apurified form (e.g., free or substantially free of polynucleotides oforigin other than the polynucleotide encoding a polypeptide with therequired function). As used herein, “substantially pure” and“substantially free,” refer to a solution or suspension containing lessthan, for example, 20% or less extraneous material, 10% or lessextraneous material, 5% or less extraneous material, 4% or lessextraneous material, 3% or less extraneous material, 2% or lessextraneous material, or 1% or less extraneous material.

A wide variety of host/expression vector combinations can be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, can consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, Pcr1, Pbr322, Pmb9 and their derivatives, plasmids such as RP4;phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2u plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Also provided herein is a recombinant host cell which comprises one ormore polynucleotide constructs. A polynucleotide encoding an antibody orantigen-binding fragment as provided herein forms an aspect of thepresent application, as does a method of production of the antibody orantigen-binding fragment which method comprises expression from thepolynucleotide. Expression can be achieved, for example, by culturingunder appropriate conditions recombinant host cells containing thepolynucleotide. An antibody or antigen-binding fragment can then beisolated and/or purified using any suitable technique, and used asappropriate.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—can beused in these vectors to express the DNA sequences. Such usefulexpression control sequences include, for example, the early or latepromoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system,the trp system, the TAC system, the TRC system, the LTR system, themajor operator and promoter regions of phage λ, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast □-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney cells, NSO mouse melanoma cells and many others. A common,bacterial host can be, for example, E. coli.

The expression of antibodies or antigen-binding fragments in prokaryoticcells, such as E. coli, is well established in the art. For a review,see for example Plückthun, A. Bio/Technology 9: 545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art (Raff, M. E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560).

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences. These hosts include well-known eukaryotic andprokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus,Streptomyces, fungi such as yeasts, and animal cells, such as CHO,YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences.Neither will all hosts function equally well with the same expressionsystem. However, one skilled in the art will be able to select theproper vectors, expression control sequences, and hosts without undueexperimentation to accomplish the desired expression without departingfrom the scope of this application. For example, in selecting a vector,the host must be considered because the vector must function in it. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, will also be considered. One of ordinary skill inthe art can select the proper vectors, expression control sequences, andhosts to accomplish the desired expression without departing from thescope of this application. For example, in selecting a vector, the hostis considered because the vector functions in it. The vector's copynumber, the ability to control that copy number, and the expression ofany other proteins encoded by the vector, such as antibiotic markers,can also be considered.

The present application also provides constructs in the form ofplasmids, vectors, transcription or expression cassettes as describedelsewhere herein which comprise at least one polynucleotide as above.Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, selectablemarkers and other sequences as appropriate. Vectors can be plasmids,viral e.g., phage, phagemid, etc., as appropriate. For further detailssee, for example, Molecular Cloning: a Laboratory Manual: 2nd edition,Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

A further aspect provides a host cell containing one or morepolynucleotides as disclosed herein. Yet a further aspect provides amethod of introducing such one or more polynucleotides into a host cell,any available technique. For eukaryotic cells, suitable techniques caninclude, for example, calcium phosphate transfection, DEAEDextran,electroporation, liposome-mediated transfection and transduction usingretrovirus or other virus (e.g. vaccinia) or, for insect cells,baculovirus. For bacterial cells, suitable techniques can include, forexample calcium chloride transformation, electroporation andtransfection using bacteriophages.

The introduction can be followed by causing or allowing expression fromthe one or more polynucleotides, e.g. by culturing host cells underconditions for expression of one or more polypeptides from one or morepolynucleotides. Inducible systems can be used and expression induced byaddition of an activator.

In one embodiment, the polynucleotides can be integrated into the genome(e.g., chromosome) of the host cell. Integration can be promoted byinclusion of sequences which promote recombination with the genome, inaccordance with standard techniques. In another embodiment, the nucleicacid is maintained on an episomal vector in the host cell.

Methods are provided herein which include using a construct as statedabove in an expression system in order to express a specificpolypeptide.

Considering these and other factors, a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences on fermentation or inlarge scale animal culture.

A polynucleotide encoding an antibody, antigen-binding fragment, or abinding protein can be prepared recombinantly/synthetically in additionto, or rather than, cloned. The polynucleotide can be designed with theappropriate codons for the antibody, antigen-binding fragment, or abinding protein. In general, one will select preferred codons for anintended host if the sequence will be used for expression. The completepolynucleotide can be assembled from overlapping oligonucleotidesprepared by standard methods and assembled into a complete codingsequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al.,Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method can be used to create analogs withunnatural amino acids.

As mentioned above, a DNA sequence encoding an antibody orantigen-binding fragment thereof can be prepared synthetically ratherthan cloned. The DNA sequence can be designed with the appropriatecodons for the antibody or antigen-binding fragment amino acid sequence.In general, one will select preferred codons for the intended host ifthe sequence will be used for expression. The complete sequence isassembled from overlapping oligonucleotides prepared by standard methodsand assembled into a complete coding sequence. See, e.g., Edge, Nature,292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J.Biol. Chem., 259:6311 (1984), each of which is which is incorporatedherein by reference in its entirety.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response, particularly to an antigen. An adjuvant can serve as atissue depot that slowly releases the antigen and also as a lymphoidsystem activator that non-specifically enhances the immune response(Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: MenloPark, Calif., p. 384). Often, a primary challenge with an antigen alone,in the absence of an adjuvant, will fail to elicit a humoral or cellularimmune response. Previously known and utilized adjuvants include, butare not limited to, complete Freund's adjuvant (CFA), incompleteFreund's adjuvant (IFA), saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, keyholelimpet hemocyanins, dinitrophenol, and potentially useful human adjuvantsuch as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.Mineral salt adjuvants include but are not limited to: aluminumhydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide andcalcium hydroxide. Preferably, the adjuvant composition furthercomprises a lipid of fat emulsion comprising about 10% (by weight)vegetable oil and about 1-2% (by weight) phospholipids. Preferably, theadjuvant composition further optionally comprises an emulsion formhaving oily particles dispersed in a continuous aqueous phase, having anemulsion forming polyol in an amount of from about 0.2% (by weight) toabout 49% (by weight), optionally a metabolizable oil in anemulsion-forming amount of up to 15% (by weight), and optionally aglycol ether-based surfactant in an emulsion-stabilizing amount of up toabout 5% (by weight).

Antibodies can also be affinity matured using known selection and/ormutagenesis methods as described above. Affinity matured antibodies canhave an affinity which is two times, five times, 10 times, 20 times, 30times or more greater than the starting antibody (generally murine,rabbit, chicken, humanized or human) from which the matured antibody isprepared. Apparent affinities can be determined by methods such as anenzyme linked immunosorbent assay (ELISA) or any other techniquefamiliar to one of skill in the art. Avidities can be determined bymethods such as a Scatchard analysis or any other technique familiar toone of skill in the art. Another technique for measuring apparentbinding affinity familiar to those of skill in the art is a surfaceplasmon resonance technique (analyzed on a BIACORE 2000 system)(Liljeblad, et al., Glyco. J. 2000, 17:323-329). Standard measurementsand traditional binding assays are described by Heeley, R. P., Endocr.Res. 2002, 28:217-229.

In one embodiment, an antibody specifically and selectively binds to themature or active form of LOX after proteolytic processing, with agreater binding affinity (e.g., at least about 5 times, at least about10 times, at least about 50 times, at least about 100 times, at leastabout 500 times, or at least about 1000 times greater), than the bindingaffinity to at least one of: the preproprotein of human LOX, thesecreted human LOX, or other lysyl oxidase-like or lysyl oxidase-relatedproteins (e.g., LOXL1, LOXL2, LOXL3, and LOXL4). In one embodiment, theantibody specifically and selectively binds to LOX in unprocessed and/orprocessed (mature) forms. The mature form of LOX is typically activealthough, in some embodiments, unprocessed LOX is also active.

In another embodiment, an antibody specifically and selectively binds tothe secreted form of LOX, such as a secreted human LOX after cleavage ofthe signal peptide, with a greater binding affinity (e.g., at leastabout 5 times, at least about 10 times, at least about 50 times, atleast about 100 times, at least about 500 times, or at least about 1000times greater), than the binding affinity to at least one of: thepreproprotein of human LOX, the mature or active human LOX, or otherlysyl oxidase-like or lysyl oxidase-related proteins (e.g., LOXL1,LOXL2, LOXL3, and LOXL4).

In yet another embodiment, an antibody specifically and selectivelybinds to LOXL2 with a greater binding affinity (e.g., at least about 5times, at least about 10 times, at least about 50 times, at least about100 times, at least about 500 times, or at least about 1000 timesgreater) than the binding affinity to at least one of: human LOX, themature or active human LOX, the secreted form of LOX or other lysyloxidase-like (LOL) or lysyl oxidase-related proteins (e.g., LOXL1,LOXL3, and LOXL4) in unprocessed, mature, active and/or secretedproducts. In one embodiment, the antibody specifically and selectivelybinds to LOXL2 in unprocessed and/or processed (mature) forms. Themature form of LOXL2 is typically active although, in some embodiments,unprocessed LOXL2 is also active.

An antibody can bind to both human LOX or human LOXL2, with a greaterbinding affinity (e.g., at least about 5 times, at least about 10 times,at least about 50 times, at least about 100 times, at least about 500times, or at least about 1000 times greater), than the binding affinityto at least one of: other lysyl oxidase-like or lysyl oxidase-relatedproteins (e.g., LOXL1, LOXL3, and LOXL4).

Antibodies that bind to enzymes can be competitive inhibitors,uncompetitive inhibitors or non-competitive inhibitors. With respect tocompetitive inhibition, an inhibitor usually bears structural similarityto substrate. Inhibition will be noticeable at low substrateconcentrations, but can be overcome at high substrate concentrations.With respect to uncompetitive inhibition, an inhibitor binds at sitethat becomes available after substrate is bound at the active site.Inhibition will be most noticeable at high substrate concentration. Withrespect to non-competitive inhibition, an inhibitor binds at site awayfrom substrate binding site. Relative inhibition will generally be thesame at all substrate concentrations. The mechanism of action antibodiesthat act as competitive inhibitors, uncompetitive inhibitors andnon-competitive inhibitors is illustrated in FIG. 4. Antibodiesdescribed herein can be competitive inhibitors, uncompetitive inhibitorsor non-competitive inhibitors. In one aspect, antibodies describedherein can be non-competitive inhibitors.

In one aspect, antibodies described herein are non-competitiveinhibitors; that is the antibodies block enzymatic activity of LOX orLOXL2 regardless of whether or not the enzymes are bound to substrate(collagen).

Binding of an antibody to LOX/LOXL2 can (1) reduce or inhibit uptake orinternalization of LOX/LOXL2 (e.g., via integrin beta 1 or othercellular receptors or proteins) and/or (2) reduce or inhibit theenzymatic activity of LOX or LOXL2. It is believed that such an antibodycould reduce EMT and thus is useful for the applications disclosedherein. An antibody described herein can bind to the proteolyticcleavage site of LOX or LOXL2, thereby effectively blocking (inhibiting)processing of the LOX or LOXL2 to reduce the level of active LOX orLOXL2. Such inhibition can occur through direct binding to LOX or LOXL2or through indirect interference including steric hindrance, enzymaticalteration of LOX or LOXL2, inhibition of transcription or translation,destabilization of mRNA transcripts, impaired export, processing, orlocalization of LOX or LOXL2, and the like.

Binding of LOX/LOXL2 with other proteins, such as cellular receptors(e.g., uptake receptor integrin beta1), BTK (burton agammagloublinemiatyrosine kinase), or other integrins is also performed using theaforementioned assay, wherein instead of ECM proteins, cellularreceptors (e.g., uptake receptor integrin beta1), BTK (burtonagammagloublinemia tyrosine kinase), or other integrins are used.

Those anti-LOX/LOXL2 antibodies that inhibit LOX/LOXL2 binding to ECMproteins, cellular receptors, and integrins, are selected as candidatesfor further development. In one embodiment, anti-LOX/LOXL2 antibodiesthat inhibit LOX/LOXL2 binding to ECM proteins, cellular receptors, andintegrins, are non-competitive inhibitors.

In one embodiment, an antibody described herein specifically binds tothe catalytic domain of LOX. This domain, in the C-terminal region,contains the elements required for catalytic activity (the copperbinding site, tyrosyl and lysyl residues that contribute to the carbonylcofactor, and 10 cysteine residues. See, Thomassin et al. “ThePro-regions of lysyl oxidase and lysyl oxidase-like 1 are required fordeposition onto elastic fibers,” J Biol. Chem. 2005 Dec. 30;280(52):42848-55 for further details.

Provided herein are antibodies or antigen binding fragments thereof thatbind to LOX and/or inhibit the activity of LOX. Anti-LOX antibodies andantigen binding fragments thereof that bind and/or inhibit LOX have usein the purification, diagnostic, and therapeutic methods describedherein. Antibodies can bind both full-length and/or processed LOX/LOXL2.

Provided herein is an isolated antibody or antigen binding fragmentthereof, comprising a variable heavy chain having at least 75% aminoacid sequence identity to an amino acid sequence set forth as SEQ ID NO:3 and a variable light chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 4 or 5.

In one aspect, provided herein is an isolated antibody or antigenbinding fragment thereof, comprising a variable heavy chain having atleast 75% amino acid sequence identity to an amino acid sequence setforth as SEQ ID NO: 3. In another aspect, provided herein is an isolatedantibody or antigen binding fragment thereof, comprising a variablelight chain having at least 75% amino acid sequence identity to an aminoacid sequence set forth as SEQ ID NO: 4 or 5.

Further provided herein are antibodies or antigen binding fragmentsthereof, that compete with, or specifically bind to, an anti-LOXantibody or antigen binding fragment thereof described herein forbinding to LOX. In one embodiment, anti-LOX antibodies described hereinare non-competitive inhibitors.

Any of such antibodies or antigen binding fragments can specificallybind to LOX with a binding affinity of at least 2, 5, 10, 50, 100, 500or 1000 times greater than to at least one of LOXL1, LOXL2, LOXL3 orLOXL4.

In one embodiment, an antibody or antigen binding fragment thereof,described herein specifically binds both full-length and processed LOX.In one aspect, both full-length and processed LOX are active forms ofthe enzyme.

Provided herein are antibodies or antigen binding fragments thereof thatbind to LOXL2 and/or inhibit the activity of LOXL2. Anti-LOXL2antibodies and antigen binding fragments thereof that bind and/orinhibit LOXL2 have use in the purification, diagnostic, and therapeuticmethods described herein.

Provided herein is an isolated antibody or antigen binding fragmentthereof, that specifically binds to an epitope having an amino acidsequence set forth as SEQ ID NO: 6. The antibody or antigen bindingfragment thereof, can comprise a variable heavy chain having at least75% amino acid sequence identity to an amino acid sequence set forth asSEQ ID NO: 1 and a variable light chain having at least 75% amino acidsequence identity to an amino acid sequence set forth as SEQ ID NO: 2.

Also provided herein is an isolated antibody or antigen binding fragmentthereof, comprising a variable heavy chain having at least 75% aminoacid sequence identity to an amino acid sequence set forth as SEQ ID NO:1, and a variable light chain having at least 75% amino acid sequenceidentity to an amino acid sequence set forth as SEQ ID NO: 2.

In one aspect, provided herein is a variable heavy chain having at least75% amino acid sequence identity to an amino acid sequence set forth asSEQ ID NO: 1. In another aspect, provided herein is a variable lightchain having at least 75% amino acid sequence identity to an amino acidsequence set forth as SEQ ID NO: 2.

Further provided herein are antibodies or antigen binding fragments thatcompete with, or specifically bind to, any of the preceding antibodiesor antigen binding fragments thereof for binding to LOXL2.

Any of such antibodies or antigen binding fragments can specificallybind to LOXL2 with a binding affinity of at least 2, 5, 10, 50, 100, 500or 1000 times greater than to at least one of LOX, LOXL1, LOXL3 orLOXL4.

In one embodiment, an antibody or antigen binding fragment thereof,described herein specifically binds to the SRCR3-4 region of LOXL2 and,thus, binds both full-length and processed LOXL2. In one aspect, bothfull-length and processed LOXL2 are active forms of the enzyme. Anantibody can, for example, specifically bind to an epitope having anamino acid sequence set forth as SEQ ID NO: 6. Such antibodies can serveas an uncompetitive partial inhibitor of enzymatic activity in vitro,inhibiting approximately half the enzymatic activity against a1,5-diaminopentane substrate with an apparent IC₅₀ of 20-30 nM. Suchantibodies can serve as non-competitive inhibitors.

When humanizing antibodies, simultaneous incorporation of all of the FRand/or CDR encoding nucleic acids and all of the selected amino acidposition changes can be accomplished by a variety of methods known tothose skilled in the art, including for example, recombinant andchemical synthesis. For example, simultaneous incorporation can beaccomplished by, for example, chemically synthesizing the nucleotidesequence for the acceptor variable region, fused together with the donorCDR encoding nucleic acids, and incorporating at the positions selectedfor harboring variable amino acid residues a plurality of correspondingamino acid codons.

Provided herein are antibodies and antigen-binding fragments thereofthat bind to LOX or LOXL2. Antibodies and antigen-binding fragmentsthereof that bind LOX or LOXL2 can inhibit (partially or fully) ormanage/treat (partially or fully) symptoms associated with and/or causedby aberrant LOX or LOXL2 expression. The application also provides celllines which can be used to produce the antibodies, methods for producingthe cell lines, methods for expressing antibodies or antigen-bindingfragments and purifying the same.

One can recognize that the antibodies and antigen-binding fragmentsthereof that specifically bind LOX or LOXL2 generated using the methodsdescribed herein can be tested using the assays provided herein or knownin the art for the ability to bind to LOX or LOXL2 (e.g., ELISA) as wellas affinity (e.g., Biacore or Surface Plasmon Resonance).

Humanized versions of anti-LOX and anti-LOXL2 antibodies have one oremore of the following characteristics: retention of the inhibitoryfunction of murine monoclonal antibodies, equivalent or increasedbinding affinity with a slow off rate (e.g., Kd 0.1-1 nM), binding tofull length and/or processed LOX/LOXL2, non-competitive partialinhibition of enzymatic activity, equivalent or better Ic50 (e.g., about30 nM), inhibitory activity in cell-based migration/invasion assays,inhibition of an EMT-like change induced by secreted LOX/LOXL2 inconditioned media of tumor cells, binding to matrix-associated LOX/LOXL2generated by live human tumor cells, cross-reactivity of binding ofhuman LOX/LOXL2 with murine LOX/LOXL2, therapeutic effectiveness (e.g.,partial or reduction in tumor size and/or symptoms), reduced toxicityand reduced immunogenicity.

Provided herein are humanized antibodies that bind to hLOX, humanizedantibodies that bind to hLOX and mLOX (murine LOX), humanized antibodiesthat bind to hLOXL2, and humanized antibodies that bind to hLOXL2 andmLOXL2 (murine LOXL2). In one aspect, the humanized antibodies arenon-competitive inhibitors.

In one embodiment, a humanized anti-LOXL2 antibody has VH chain havingan amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 orSEQ ID NO: 28. One would understand that conservative amino acidmodifications can be made using the methods described herein in one ormore CDR or framework regions for affinity maturation of the antibody.Antibodies modified by such methods can be tested with respect tofunction using any of the assays described herein or known in the art.

In another embodiment, a humanized anti-LOXL2 antibody has VL chainhaving an amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31 or SEQ IDNO: 32. One would understand that conservative amino acid modificationscan be made using the methods described herein in one or more CDR orframework regions for affinity maturation of the antibody. Antibodiesmodified by such methods can be tested with respect to function usingany of the assays described herein or known in the art.

Provided herein is a humanized antibody, or antigen-binding fragmentthereof, which binds LOXL2, comprising a heavy chain variable region anda light chain variable region,

wherein said heavy chain variable region comprises:

-   -   (i) a heavy chain FR1 having the amino acid sequence of SEQ ID        NO: 33 or the amino acid sequence of SEQ ID NO: 33 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of glutamine (Q) by valine (V) or a            conservative substitution thereof at position 24;        -   (b) a substitution of leucine (L) by valine (V) or a            conservative substitution thereof at position 30;        -   (c) a substitution of valine (V) by lysine (K) or a            conservative substitution thereof at position 31;        -   (d) a substitution of arginine (R) by lysine (K) or a            conservative substitution thereof at position 32; and        -   (e) a substitution of threonine (T) by alanine (A) or a            conservative substitution thereof at position 35;        -   and a deletion of amino acid residues 1-19;    -   (ii) a heavy chain FR2 having the amino acid sequence of SEQ ID        NO: 34 or the amino acid sequence of SEQ ID NO: 34 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of lysine (K) by arginine (R) or a            conservative substitution thereof at position 3;        -   (b) a substitution of arginine (R) by alanine (A) or a            conservative substitution thereof at position 5, and    -   (iii) a heavy chain FR3 having the amino acid sequence of SEQ ID        NO: 35 or the amino acid sequence of SEQ ID NO: 35 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of lysine (K) by arginine (R) or a            conservative substitution thereof at position 1;        -   (b) a substitution of alanine (A) by valine (V) or a            conservative substitution thereof at position 2;        -   (c) a substitution of leucine (L) by isoleucine (I) or a            conservative substitution thereof at position 4;        -   (d) a substitution of serine (S) by threonine (T) or a            conservative substitution thereof at position 10;        -   (e) a substitution of glutamine (Q) by glutamic acid (E) or            a conservative substitution thereof at position 16;        -   (f) a substitution of threonine (T) by arginine (R) or a            conservative substitution thereof at position 21;        -   (g) a substitution of aspartic acid (D) by glutamic acid (E)            or a conservative substitution thereof at position 23;        -   (h) a substitution of serine (S) by threonine (T) or a            conservative substitution thereof at position 25; and        -   (i) a substitution of phenylalanine (F) by tyrosine (Y) or a            conservative substitution thereof at position 29;        -   and    -   (iv) a heavy chain FR4 having the amino acid sequence of SEQ ID        NO: 36 or the amino acid sequence of SEQ ID NO: 36 but for a        substitution of lysine (K) by valine (V) or a conservative        substitution thereof at position 7,    -   and wherein said light chain variable region comprises:    -   (i) a light chain FR1 having the amino acid sequence of SEQ ID        NO: 49 or the amino acid sequence of SEQ ID NO: 49 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of alanine (A) by threonine (T) or a            conservative substitution thereof at position 27;        -   (b) a substitution of alanine (A) by proline (P) or a            conservative substitution thereof at position 28;        -   (c) a substitution of proline (P) by leucine (L) or a            conservative substitution thereof at position 29;        -   (d) a substitution of valine (V) by leucine (L) or a            conservative substitution thereof at position 31;        -   (e) a substitution of glutamic acid (E) by glutamine (Q) or            a conservative substitution thereof at position 37;        -   (f) a substitution of valine (V) by alanine (A) or a            conservative substitution thereof at position 39;        -   and a deletion of amino acid residues 1-20;    -   (ii) a light chain FR2 having the amino acid sequence of SEQ ID        NO: 50 or the amino acid sequence of SEQ ID NO: 50 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of phenylalanine (F) by tyrosine (Y) or a            conservative substitution thereof at position 2; and        -   (b) a substitution of arginine (R) by lysine (K) or a            conservative substitution thereof at position 5;    -   (iii) a light chain FR3 having the amino acid sequence of SEQ ID        NO: 51 or the amino acid sequence of SEQ ID NO: 51 but for one        or more substitutions selected from the group consisting of:        -   (a) a substitution of alanine (A) by aspartic acid (D) or a            conservative substitution thereof at position 14; and        -   (b) a substitution of arginine (R) by lysine (K) or a            conservative substitution thereof at position 18;    -   and    -   (iv) a light chain FR4 having the amino acid sequence of SEQ ID        NO: 52 or the amino acid sequence of SEQ ID NO: 52 but for a        substitution of leucine (L) by valine (V) or a conservative        substitution thereof at position 7;

In one embodiment, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region FR1 having an amino acidsequence as set forth in SEQ ID NO: 33, 37 or 44; a heavy chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 34,38 or 45; a heavy chain variable region FR3 having an amino acidsequence as set forth in SEQ ID NO: 35, 39, 46, 47 or 48; a heavy chainvariable region FR4 having an amino acid sequence as set forth in SEQ IDNO: 36 or 40; a light chain variable region FR1 having an amino acidsequence as set forth in SEQ ID NO: 49 or 53; a light chain variableregion FR2 having an amino acid sequence as set forth in SEQ ID NO: 50,54 or 60; a light chain variable region FR3 having an amino acidsequence as set forth in SEQ ID NO: 51, 55 or 61; and a light chainvariable region FR4 having an amino acid sequence as set forth in SEQ IDNO: 52 or 56.

Encompassed within the scope of the present application are variableheavy chains and variable light chains that are at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95% or up to 100% identical to the variable heavychains and variable light chains described herein.

Conservative substitutions are minor modification of these nucleotidesequences and/or amino acids are intended to be included as heavy andlight chain encoding nucleic acids and their functional fragments. Suchminor modifications include, for example, those which do not change theencoded amino acid sequence due to the degeneracy of the genetic code aswell as those which result in only a conservative substitution of theencoded amino acid sequence or those that do not substantially alter thebinding capacity of the antibody. Conservative substitutions of encodedamino acids include, for example, amino acids which belong within thefollowing groups: (1) non-polar amino acids (Gly, Ala, Val, Leu, andIle); (2) polar neutral amino acids (Cys, Met, Ser, Thr, Asn, and Gln);(3) polar acidic amino acids (Asp and Glu); (4) polar basic amino acids(Lys, Arg and His); and (5) aromatic amino acids (Phe, Trp, Tyr, andHis). Other minor modifications are included within the nucleic acidsencoding heavy and light chain polypeptides of the invention so long asthe nucleic acid or encoded polypeptides retain some, or all, of theirfunction as described herein and which have use in the methods describedherein. Non-conservative substitutions are those that are not identifiedas conservative substitutions. Using the methods described herein, onecan ascertain whether it would be possible to substitute anon-conservative amino acid for a framework amino acid residue and testthe function of the modified antibody using the assays describedelsewhere herein.

Modified variable heavy chains and variable light chains can be screenedfor binding and activity using methods known in the art and describedherein.

A substantial portion of a variable domain will include three CDRregions, together with their intervening framework regions. The portioncan also include at least about 50% of either or both of the first andfourth framework regions, the 50% being the C-terminal 50% of the firstframework region and the N-terminal 50% of the fourth framework region.Additional residues at the N-terminal or C-terminal end of thesubstantial part of the variable domain may be those not normallyassociated with naturally occurring variable domain regions. Forexample, construction of humanized anti-LOX or anti-LOXL2 antibodies andantigen-binding fragments described herein made by recombinant DNAtechniques can result in the introduction of N- or C-terminal residuesencoded by linkers introduced to facilitate cloning or othermanipulation steps. Other manipulation steps include the introduction oflinkers to join variable domains to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailbelow.

Antibodies encompassed within the present application, including, forexample, those having variable heavy or light chains that have at least50% identity to those described herein can be assessed for anti-LOX oranti-LOXL2 activity.

Provided herein is a method for identifying an antibody that inhibitsmetastatic tumor cell growth, comprising contacting LOX or LOXL2 or acell expressing LOX or LOXL2 with a candidate antibody; and determiningthe expression or activity of the LOX or LOXL2, whereby the candidateantibody that reduces the expression or activity of LOX or LOXL2compared to the expression or activity detected in the absence of theantibody is identified as the compound that inhibits metastatic tumorcell growth. In particular embodiments, the antibody is contacted withLOX or LOXL2 or a cell expressing LOX or LOXL2 under hypoxic conditions.In one aspect, antibodies described herein can be non-competitiveinhibitors.

Also provided herein is method for identifying an antibody thatincreases the efficacy of chemotherapeutic agents, comprising contactingLOX or LOXL2 or a cell expressing LOX or LOXL2 with a candidateantibody; and determining the expression or activity of the LOX orLOXL2, whereby the candidate antibody that reduces the expression oractivity of LOX or LOXL2 compared to the expression or activity detectedin the absence of the antibody is identified as the antibody thatincreases the efficacy of chemotherapeutic agents in inhibiting orreducing metastatic tumor growth.

Any suitable source of LOX or LOXL2 can be employed as an antibodytarget in the present method. The enzyme can be derived, isolated, orrecombinantly produced from any source known in the art, includingyeast, microbial, and mammalian, that will permit the generation of asuitable product that can generate a detectable reagent or will bebiologically active in a suitable assay.

The enzymatic activity of LOX or LOXL2 can be assessed by any suitablemethod described herein or known in the art. Exemplary methods ofassessing LOX or LOXL2 activity include that of Trackman et al., Anal.Biochem. 113:336-342 (1981); Kagan, et al., Methods Enzymol. 82A:637-49(1982); Palamakumbura et al., Anal. Biochem. 300:245-51 (2002); Albiniet al., Cancer Res. 47: 3239-45 (1987); Kamath et al, Cancer Res.61:5933-40 (2001); U.S. Pat. No. 4,997,854; and U.S. Patent ApplicationNo. 2004/0248871. For example, enzymatic activity can be assessed bydetecting and/or quantitating “lysyl oxidase byproducts,” such as H₂O₂production; collagen pyridinium residues, ammonium production; aldehydeproduct production; lysyl oxidation, deoxypyridinoline (Dpd)—discussedbelow. One may also detect and quantitate cellular invasive capacity invitro; cellular adhesion and growth in vitro; and metastatic growth invivo. In vivo models include, but are not limited to suitable syngeneicmodels, human tumor xenograft models, orthotopic models, metastaticmodels, transgenic models, and gene knockout models (see, e.g., Teicher,Tumors Models in Cancer Research (Humana Press 2001)).

Hypoxic conditions can be induced or naturally occurring. Hypoxic areasfrequently occur in the interior of solid tumor. Hypoxia can also beinduced in vivo, particularly in experimental animal models, usingdiminution or cessation of arterial blood flow to tumor or theadministration of vasoconstrictive compounds. See, e.g., U.S. Pat. No.5,646,185. Exemplary vasoconstrictive compounds include adrenergicdirect and indirect agonists such as norepinephrine, epinephrine,phenylephrine, and cocaine. The presence of a hypoxic region in a solidtumor present in a subject can be observed by a number of methodscurrently known in the art, including nuclear magnetic resonance (NMR)and oxygen electrode pO₂ histography. Such methods may be used in thecontext of the present invention (as described below), to identifyhypoxic treatment target regions and to guide in administering treatmentcompositions to such regions. In vitro, hypoxic conditions can beinduced using any suitable method. For example, cells can be maintainedunder anoxic (<0.1% O₂) conditions at 37° C. within an anaerobic chamberor under hypoxic (1 to 2% O₂) conditions at 37° C. within a modularincubator chamber filled with 5% CO₂ and 1 to 2% O₂ balanced with N₂.See, e.g., Erler et al., Mol. Cell. Biol. 24:2875-89 (2004).

The LOX or LOXL2 enzymes or LOX- or LOXL2-expressing cell can becontacted with a compound (e.g., a LOX/LOXL inhibitor such as anantibody) in any suitable manner for any suitable length of time. Fortumor regions that are accessible to hypodermic delivery of agent, itmay be desirable to inject the compound directly into the hypoxicregion. The cells can be contacted with the compound more than onceduring incubation or treatment. Typically, the dose required for anantibody is in the range of about 1 micro-g/ml to 1000 micro-g/ml, moretypically in the range of about 100 μg/ml to about 800 μg/ml. The exactdose can be readily determined from in vitro cultures of the cells andexposure of the cell to varying dosages of the compound. Typically, thelength of time the cell is contacted with the compound is about 5minutes, about 15 minutes, about 30 minutes, about 1 hour, about 4hours, about 12 hours, about 36 hours, about 48 hours to about 3 days ormore, even indefinitely, more typically for about 24 hours. For in vitroinvasion assays, any suitable matrix may be used. In one embodiment, thematrix is reconstituted basement membrane Matrigel™ matrix (BDSciences).

Screening methods can also include a step of measuring FAK levels. Asdescribed below, FAK (Focal Adhesion Kinase [p125FAK]) is activated aspart of the cell motility process. When LOX is inhibited, FAKphosphorylation is not increased under hypoxic conditions. In acompound-screening assay, a secondary step can include the detection ofphospho-FAK levels both with and without addition of the test antibody.A test inhibitory antibody will also reduce levels of phospho-FAK.

An antibody is an inhibitor of LOX or LOXL2 expression or biologicalactivity when the antibody reduces the expression or activity or LOX orLOXL2 relative to that observed in the absence of the antibody. In oneembodiment, an antibody is an inhibitor of LOX or LOXL2 when it reducesthe incidence of metastasis relative to the observed in the absence ofthe antibody and, in further testing, inhibits metastatic tumor growth.In one aspect, antibodies described herein are non-competitiveinhibitors. Tumor inhibition can be quantified using any convenientmethod of measurement. The incidence of metastasis can be assessed byexamining relative dissemination (e.g., number of organ systemsinvolved) and relative tumor burden in these sites. Metastatic growthcan be ascertained by microscopic or macroscopic analysis, asappropriate. Tumor metastasis can be reduced by about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or greater. In some embodiments, theantibody can be assessed relative to other antibodies or compounds thatdo not impact LOX or LOXL2 expression or biological activity. The testantibodies can be administered at the time of tumor inoculation, afterthe establishment of primary tumor growth, or after the establishment oflocal and/or distant metastases. Single or multiple administration ofthe test antibody can be given using any convenient mode ofadministration including, but not limited to, intravenous,intraperitoneal, intratumoral, subcutaneous and intradermal.

Any suitable cell expressing LOX or LOXL2 can be employed with thepresent methods. As used herein, the term “cell” includes a biologicalcell (e.g., CHO, HeLa, etc.). The cell can be human or nonhuman. Thecell can be freshly isolated (i.e., primary) or derived from a shortterm- or long term-established cell line. Exemplary biological celllines include MDA-MB 231 human breast cancer cells, MDA-MB 435 humanbreast cancer cells, U-87 MG glioma, SCL1 squamous cell carcinoma cells,CEM, HeLa epithelial carcinoma, and Chinese hamster ovary (CHO) cells.Such cell lines are described, for example, in the Cell Line Catalog ofthe American Type Culture Collection (ATCC, Rockville, Md.).

A cell can express the LOX or LOXL2 or its promoter endogenously orexogenously (e.g., as a result of the stable transfer of genes).Endogenous expression by a cell as provided herein can result fromconstitutive or induced expression of endogenous genes.

Exogenous expression by a cell as provided herein can result from theintroduction of the nucleic acid sequences encoding LOX or LOXL2 or abiologically active fragment thereof, or LOX or LOXL2 promoter nucleicacid sequence. Transformation may be achieved using viral vectors,calcium phosphate, DEAE-dextran, electroporation, biolistics, cationiclipid reagents, or any other convenient technique known in the art. Themanner of transformation useful in the present invention is conventionaland is exemplified in Current Protocols in Molecular Biology (Ausubel,et al., eds. 2000). Exogenous expression of the lysyl oxidase or itspromoter can be transient, stable, or some combination thereof.Exogenous expression of the enzyme can be achieved using constitutivepromoters, e.g., SV40, CMV, and the like, and inducible promoters knownin the art. Suitable promoters are those that will function in the cellof interest.

The methods described herein are non-limiting and any other methodsknown in the art can also be used to test the activity of anti-LOXantibodies and anti-LOXL2 antibodies. Additional assays are describedbelow in the Examples.

It may be necessary in some instances to introduce an unstructuredpolypeptide linker region between a label of the present invention andportions of the antibodies. The linker can facilitate enhancedflexibility, and/or reduce steric hindrance between any two fragments.The linker can also facilitate the appropriate folding of each fragmentto occur. The linker can be of natural origin, such as a sequencedetermined to exist in random coil between two domains of a protein. Anexemplary linker sequence is the linker found between the C-terminal andN-terminal domains of the RNA polymerase a subunit. Other examples ofnaturally occurring linkers include linkers found in the ICI and LexAproteins.

Within the linker, the amino acid sequence may be varied based on thepreferred characteristics of the linker as determined empirically or asrevealed by modeling. Considerations in choosing a linker includeflexibility of the linker, charge of the linker, and presence of someamino acids of the linker in the naturally-occurring subunits. Thelinker can also be designed such that residues in the linker contactDNA, thereby influencing binding affinity or specificity, or to interactwith other proteins. In some cases, particularly when it is necessary tospan a longer distance between subunits or when the domains must be heldin a particular configuration, the linker may optionally contain anadditional folded domain.

In some embodiments it is preferable that the design of a linker involvean arrangement of domains which requires the linker to span a relativelyshort distance, preferably less than about 10 Angstroms (Å). However, incertain embodiments, linkers span a distance of up to about 50 Å.

Antibodies provided herein such that they are conjugated or linked totherapeutic and/or imaging/detectable moieties. Methods for conjugatingor linking antibodies are well known in the art. Associations betweenantibodies and labels include any means known in the art including, butnot limited to, covalent and non-covalent interactions.

In one non-limiting embodiment, antibodies can be associated with atoxin, a radionuclide, an iron-related compound, a dye, an imagingreagent, a fluorescent label or a chemotherapeutic agent that would betoxic when delivered to a cancer cell.

Alternatively, the antibodies can be associated with detectable label,such as a radionuclide, iron-related compound, a dye, an imaging agentor a fluorescent agent for immunodetection of target antigens.

Non-limiting examples of radiolabels include, for example, ³²P, ³³P,⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga, ⁷¹Ge, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁷As,⁷⁷Br, ⁸¹Rb/^(81M)Kr, ^(87M)Sr, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ¹⁰⁰Pd, ¹⁰¹Rh, ¹⁰³Pb,¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹¹⁹Sb, ¹²¹Sn, ¹²³I, ¹²⁵I, ¹²⁷Cs,¹²⁸Ba, ¹²⁹Cs, ¹³¹I, ¹³¹Cs, ¹⁴³Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Eu, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹¹Os, ¹⁹³Pt, ¹⁹⁴Ir, ¹⁹⁷Hg, ¹⁹⁹Au, ²⁰³Pb, ²¹¹At,²¹²Pb, ²¹²Bi and ²¹³Bi.

Non-limiting examples of toxins include, for example, diphtheria Achain, nonbinding active fragments of diphtheria toxin, exotoxin A chain(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes,Clostridium perfringens phospholipase C (PLC), bovine pancreaticribonuclease (BPR), antiviral protein (PAP), abrin, cobra venom factor(CVF), gelonin (GEL), saporin (SAP) viscumin.

Non-limiting examples of iron-related compounds include, for example,magnetic iron-oxide particles, ferric or ferrous particles, Fe²⁰³ andFe³⁰⁴. Iron-related compounds and methods of labeling polypeptides,proteins and peptides can be found, for example, in U.S. Pat. Nos.4,101,435 and 4,452,773, and U.S. published applications 20020064502 and20020136693, all of which are hereby incorporated by reference in theirentirety.

In certain embodiments, the subject antibodies can be covalently ornon-covalently coupled to a cytotoxin or other cell proliferationinhibiting compound, in order to localize delivery of that agent to atumor cell. For instance, the agent can be selected from the groupconsisting agents, enzyme inhibitors, proliferation inhibitors, lyticagents, DNA or RNA synthesis inhibitors, membrane permeabilitymodifiers, DNA metabolites, dichloroethylsulfide derivatives, proteinproduction inhibitors, ribosome inhibitors, inducers of apoptosis, andneurotoxins.

In certain embodiments, the subject antibodies can be coupled with anagent useful in imaging tumors. Such agents include: metals; metalchelators; lanthanides; lanthanide chelators; radiometals; radiometalchelators; positron-emitting nuclei; microbubbles (for ultrasound);liposomes; molecules microencapsulated in liposomes or nanosphere;monocrystalline iron oxide nanocompounds; magnetic resonance imagingcontrast agents; light absorbing, reflecting and/or scattering agents;colloidal particles; fluorophores, such as near-infrared fluorophores.In many embodiments, such secondary functionality/moiety will berelatively large, e.g., at least 25 amu in size, and in many instancescan be at least 50,100 or 250 amu in size.

In certain embodiments, the secondary functionality is a chelate moietyfor chelating a metal, e.g., a chelator for a radiometal or paramagneticion. In additional embodiments, it is a chelator for a radionuclideuseful for radiotherapy or imaging procedures.

Radionuclides useful within the present invention includegamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters and fluorescence-emitters, with beta-or alpha-emitterspreferred for therapeutic use. Examples of radionuclides useful astoxins in radiation ³²P, ³³P, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga,⁷¹Ge, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁷⁷As, ⁷⁷Br, ⁸¹Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc,¹⁰⁰Pd, ¹⁰¹Rh, ¹⁰³Pb, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹¹⁹Sb, ¹²¹Sn,¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁸Ba, ¹²⁹Cs, ¹³¹I, ¹³¹Cs, ¹⁴³Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Ho, ¹⁶⁹Eu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹¹Os, ¹⁹³Pt, ¹⁹⁴Ir, ¹⁹⁷Hg,¹⁹⁹Au, ²⁰³Pb, ²¹¹At, ²¹²Pb, ²¹²Bi and ²¹³Bi. Preferred therapeuticradionuclides include ¹⁸⁸Re, ¹⁸⁶Re, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ⁷⁷Br, ²¹¹At, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁹⁸Au and ¹⁹⁹Ag, ¹⁶⁶Hoor ¹⁷⁷Lu. Conditions under which a chelator will coordinate a metal aredescribed, for example, by Gasnow et al. U.S. Pat. Nos. 4,831,175,4,454,106 and 4,472,509, each of which is incorporated herein byreference. Within the present invention, “radionuclide” and “radiolabel”are interchangeable.

⁹⁹Tc is a particularly attractive radioisotope for diagnosticapplications, as it is readily available to all nuclear medicinedepartments, is inexpensive, gives minimal patient radiation doses, andhas ideal nuclear imaging properties. It has a half-life of six hourswhich means that rapid targeting of a technetium-labeled antibody isdesirable. Accordingly, in certain preferred embodiments, the modifiedantibodies include a chelating agent for technium.

In still other embodiments, the secondary functionality can be aradiosensitizing agent, e.g., a moiety that increases the sensitivity ofcells to radiation. Examples of radiosensitizing agents includenitroimidazoles, metronidazole and misonidazole (see: DeVita, V. T. inHarrison's Principles of Internal Medicine, p. 68, McGraw-Hill Book Co.,NY, 1983, which is incorporated herein by reference). The modifiedantibodies that comprise a radiosensitizing agent as the active moietyare administered and localize at the target cell. Upon exposure of theindividual to radiation, the radiosensitizing agent is “excited” andcauses the death of the cell.

There are a wide range of moieties which can serve as chelators andwhich can be derivatized to the antibodies of the present invention. Forinstance, the chelator can be a derivative of1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA) and1-p-Isothiocyanato-benzyl-methyl-diethylenetriaminepentaacetic acid(ITC-MX). These chelators typically have groups on the side chain bywhich the chelator can be used for attachment to subject antagonists.Such groups include, e.g., benzylisothiocyanate, by which the DOTA, DTPAor EDTA can be coupled to, e.g., an amine group.

In one embodiment, the chelate moiety is an “NxSy” chelate moiety. Asdefined herein, the “NxSy chelates” include bifunctional chelators thatare capable of coordinately binding a metal or radiometal and,preferably, have N2S2 or N3S cores. Exemplary NxSy chelates aredescribed, e.g., in Fritzberg et al. (1998) PNAS 85: 4024-29; and Weberet al. (1990) Chem. 1: 431-37; and in the references cited therein.

Jacobsen et al. (PCT application WO 98/12156) provides methods andcompositions, i.e., synthetic libraries of binding moieties, foridentifying compounds which bind to a metal atom. The approach describedin that publication can be used to identify binding moieties which cansubsequently be added to the antibodies to derive the modifiedantibodies.

One problem frequently encountered with the use of conjugated proteinsin and radiodiagnostic applications is a potentially dangerousaccumulation of the radiolabeled moiety fragments in the kidney. Whenthe conjugate is formed using an acid-or base-labile linker, cleavage ofthe radioactive chelate from the protein can advantageously occur. Ifthe chelate is of relatively low molecular weight, as most of thesubject modified antibodies, antigen binding fragments and peptides areexpected to be, it is not retained in the kidney and is excreted in theurine, thereby reducing the exposure of the kidney to radioactivity.However, in certain instances, it may be advantageous to utilize acid-or base-labile in the subject ligands for the same reasons they havebeen used in labeled proteins.

Accordingly, certain of the subject labeled/modified antibodies can besynthesized, by standard methods in the art, to provide reactivefunctional groups which can form acid-labile linkages with, e.g., acarbonyl group of the ligand. Examples of suitable acid-labile linkagesinclude hydrazone and thiosemicarbazone functions. These are formed byreacting the oxidized carbohydrate with chelates bearing hydrazide,thiosemicarbazide, and functions, respectively.

Alternatively, base-cleavable which have been used for the enhancedclearance of the radiolabel from the kidneys, can be used. See, forexample, Weber et al. 1990 Bioconjg. Chem. 1:431. The coupling of abifunctional chelate to an antibody via a hydrazide linkage canincorporate base-sensitive ester moieties in a linker spacer arm. Suchan ester-containing linker unit is exemplified by ethylene glycolbis(succinimidyl succinate), (EGS, available from Pierce Chemical Co.,Rockford, Ill.), which has two terminal N-hydroxysuccinimide (NHS) esterderivatives of two 1,4-dibutyric acid units, each of which are linked toa single ethylene glycol moiety by two alkyl esters. One NHS ester maybe replaced with a suitable amine-containing BFC (for example2-aminobenzyl DTPA), while the other NHS ester is reacted with alimiting amount of hydrazine. The resulting hyrazide is used forcoupling to the antagonists, forming an ligand-BFC linkage containingtwo alkyl ester functions. Such a conjugate is stable at physiologicalpH, but readily cleaved at basic pH.

Antibodies labeled by chelation of radioisotopes are subject toradiation-induced scission of the chelator and to loss of radioisotopeby dissociation of the coordination complex. In some instances, metaldissociated from the complex can be re-complexed, providing more rapidclearance of non-specifically localized isotope and therefore lesstoxicity to non-target tissues. For example, chelator compounds such asEDTA or DTPA can be infused into patients to provide a pool of chelatorto bind released radiometal and facilitate excretion of freeradioisotope in the urine.

In still other embodiments, the antibodies are coupled to a Boronaddend, such as a carborane. For example, carboranes can be preparedwith carboxyl functions on pendant side chains, as is well known in theart. Attachment of such carboranes to amine peptides can be achieved byactivation of the carboxyl groups of the carboranes and condensationwith the amine group to produce the conjugate. Such modified antibodiescan be used for neutron capture therapy.

The present invention also contemplates the modification of the subjectantagonists with dyes, for example, useful in therapy, and used inconjunction with appropriate non-ionizing radiation. The use of lightand porphyrins in methods of the present invention is also contemplatedand their use in cancer therapy has been reviewed by van den Bergh,Chemistry in Britain, 22: 430-437 (1986), which is incorporated byreference herein in its entirety.

One embodiment of the present invention includes antagonists labeledwith a fluorescent label. Common fluorescent labels include, forexample, FITC, PE, Texas Red, cytochrome c, etc. Techniques for labelingpolypeptides and fragments thereof, such as those provided herein, arewell-known in the art.

The term “anticancer agent” also includes the chemotherapeutic agentsdescribed below. The term anticancer agent also includes treatment witha substance that reduces hypoxia in a cell, when such agent is combinedwith LOX inhibition. Such a substance may include, e.g., p53. See, e.g.,Matoba et al., “p53 Regulates Mitochondrial Respiration,” Science 16Jun. 2006 312: 1650-1653; published online 24 May 2006, and referencescited there. A substance that drives cancer cells towards therespiratory pathway and away from the glycolytic pathway would be usedadvantageously with a LOX inhibitor insofar as LOX would not beup-regulated in this case.

Chemotherapeutics useful as active moieties which when conjugated toantagonists thereof of the present invention are specifically deliveredto cells are typically, small chemical entities produced by chemicalsynthesis. Chemotherapeutics include cytotoxic and cytostatic drugs.Chemotherapeutics may include those which have other effects on cellssuch as reversal of the transformed state to a differentiated state orthose which inhibit cell replication. Examples of known cytotoxic agentsuseful in the present invention are listed, for example, in Goodman etal., “The Pharmacological Basis of Therapeutics,” Sixth Edition, A. B.Gilman et al., eds./Macmillan Publishing Co. New York, 1980. Theseinclude taxanes, such as paclitaxel and docetaxel; nitrogen such asmechlorethamine, melphalan, uracil mustard and chlorambucil;ethylenimine derivatives, such as thiotepa; alkyl sulfonates, such asbusulfan; nitrosoureas, such as lomustine, semustine and streptozocin;triazenes, such as dacarbazine; folic acid analogs, such asmethotrexate; pyrimidine analogs, such as fluorouracil, cytarabine andazaribine; purine analogs, such as mercaptopurine and thioguanine; vincaalkaloids, such as vinblastine and vincristine; antibiotics, such asdactinomycin, daunorubicin, doxorubicin, and mitomycin; enzymes, such asplatinum coordination complexes, such as cisplatin; substituted urea,such as hydroxyurea; methyl hydrazine derivatives, such as procarbazine;adrenocortical suppressants, such as mitotane; hormones and antagonists,such as adrenocortisteroids (prednisone), progestins(hydroxyprogesterone caproate, acetate and megestrol acetate), estrogens(diethylstilbestrol and ethinyl estradiol), and androgens (testosteronepropionate and fluoxymesterone).

Drugs that interfere with protein synthesis can also be used; such drugsare known to those skilled in the art and include puromycin,cycloheximide, and ribonuclease.

Most of the chemotherapeutic agents currently in use in treating cancerpossess functional groups that are amenable to chemical cross-linkingdirectly with an amine or carboxyl group of an agent of the presentinvention. For example, free amino groups are available on methotrexate,doxorubicin, daunorubicin, cytosinarabinoside, bleomycin, fludarabine,and cladribine while free carboxylic acid groups are available onmethotrexate, melphalan and chlorambucil.

These functional groups, that is free amino and carboxylic acids, aretargets for a variety of homobifunctional and heterobifunctionalchemical cross-linking agents which can crosslink these drugs directlyto a free amino group of an antagonist.

Chemotherapeutic agents contemplated by the present invention alsoinclude other chemotherapeutic drugs that are commercially available.Merely to illustrate, the chemotherapeutic can be an inhibitor ofchromatin function, a inhibitor, a inhibiting drug, a DNA damagingagent, an antimetabolite (such as folate antagonists, pyrimidineanalogs, purine analogs, and sugar-modified analogs), a DNA synthesisinhibitor, a DNA interactive agent (such as an intercalating agent), aDNA repair inhibitor.

Chemotherapeutic agents may be categorized by their mechanism of actioninto, for example, the following groups: anti-metabolites/anti-canceragents, such as pyrimidine analogs floxuridine, capecitabine, andcytarabine) and purine analogs, folate antagonists and relatedinhibitors antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloid (vinblastine, vincristine, andmicrotubule such as taxane (paclitaxel, docetaxel), vinblastin,nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide,teniposide), DNA damaging agents (actinomycin, amsacrine, busulfan,carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan,dactinomycin, daunorubicin, doxorubicin, epirubicin, iphosphamide,melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide; antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards cyclophosphamide andanalogs, melphalan, chlorambucil), and (hexamethylmelamine andthiotepa), alkyl nitrosoureas (BCNU) and analogs, streptozocin),trazenes-dacarbazinine (DTIC); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate); platinumcoordination complexes (cisplatin, oxiloplatinim, carboplatin),procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones,hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel; antimigratory agents; antisecretory agents (breveldin);immunosuppressives tacrolimus sirolimus azathioprine, mycophenolate;compounds (TNP-470, genistein) and growth factor inhibitors (vascularendothelial growth factor inhibitors, fibroblast growth factorinhibitors); angiotensin receptor blocker, nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab, rituximab); cellcycle inhibitors and differentiation inducers (tretinoin); inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), daunorubicin,dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecanand mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone,dexamethasone, hydrocortisone, methylpednisolone, prednisone, andprenisolone); growth factor signal transduction kinase inhibitors;dysfunction inducers, toxins such as Cholera toxin, ricin, Pseudomonasexotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheriatoxin, and caspase activators; and chromatin. Preferred dosages of thechemotherapeutic agents are consistent with currently prescribeddosages.

Additionally, other labels, such as biotin followed bystreptavidin-alkaline phosphatase (AP), horseradish peroxidase arecontemplated by the present invention.

As used herein, the terms “nucleic acid damaging treatment” and “nucleicacid damaging agent” refer to any treatment regimen that directly orindirectly damages nucleic acid (e.g., DNA, cDNA, genomic DNA, mRNA,tRNA or rRNA). Examples of such agents include alkylating agents,nitrosoureas, anti-metabolites, plant alkaloids, plant extracts andradioisotopes. Examples of agents also include nucleic acid damagingdrugs, for example, 5-fluorouracil (5-FU), capecitabine, S-1 (Tegafur,5-chloro-2,4-dihydroxypyridine and oxonic acid), 5-ethynyluracil,arabinosyl cytosine (ara-C), 5-azacytidine (5-AC),2′,2′-difluoro-2′-deoxycytidine (dFdC), purine antimetabolites(mercaptopurine, azathiopurine, thioguanine), gemcitabine hydrochloride(Gemzar), pentostatin, allopurinol, 2-fluoro-arabinosyl-adenine(2F-ara-A), hydroxyurea, sulfur mustard (bischloroetyhylsulfide),mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide,thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromoducitol, alkylsulfonate (busulfan), nitrosoureas (BCNU, CCNU, 4-methyl CCNU or ACNU),procarbazine, decarbazine, rebeccamycin, anthracyclins such asdoxorubicin (adriamycin; ADR), daunorubibcin (Cerubicine), idarubicin(Idamycin) and epirubicin (Ellence), anthracyclin analogues such asmitoxantrone, actinomycin D, non intercalating topoisomerase inhibitorssuch as epipodophyllotoxins (etoposide=VP16, teniposide=VM-26),podophylotoxin, bleomycin (Bleo), pepleomycin, compounds that formadducts with nucleic acid including platinum derivatives (e.g.,cisplatin (CDDP), trans analogue of cisplatin, carboplatin, iproplatin,tetraplatin and oxaliplatin), camptothecin, topotecan, irinotecan(CPT-11), and SN-38. Specific examples of nucleic acid damagingtreatments include radiation (e.g., focused microwaves, ultraviolet(UV), infrared (IR), or alpha-, beta- or gamma-radiation) andenvironmental shock (e.g., hyperthermia).

As used herein, the terms “anti-proliferative treatment” and“anti-proliferative agent” means any treatment regimen that directly orindirectly inhibits proliferation of a cell, virus, bacteria or otherunicellular or multicellular organism regardless of whether or not thetreatment or agent damages nucleic acid. Particular examples ofanti-proliferative agents are anti-tumor and anti-viral drugs, whichinhibit cell proliferation or virus proliferation or replication.Examples include, inter alia, cyclophosphamide, azathioprine,cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine,busulphan, methotrexate, 6-mercaptopurine, thioguanine, cytosinearabinoside, taxol, vinblastine, vincristine, doxorubicin, actinomycinD, mithramycin, carmustine, lomustine, semustine, streptozotocin,hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine anddibromomannitol. Anti proliferative agents that cause nucleic acidreplication errors or inhibit nucleic acid replication are those such asnucleoside and nucleotide analogues (e.g., AZT or 5-AZC).

In another embodiment, the anti-LOX antibody can be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionuclide).

Methodology for labeling polypeptides and fragments thereof including,but not limited to, those provided herein are well known in the art.When the antibodies of the present invention are labeled with aradiolabel or toxin, the antibodies can be prepared as pharmaceuticalcompositions which are useful for therapeutic treatment of patientswhere the pharmaceutical compositions are administered to the patient inan effective amount. When the antibodies of the present invention arelabeled with a label that can be visualized, the antibodies can beprepared as pharmaceutical compositions which are useful for diagnosticof patients where the pharmaceutical compositions are administered tothe patient in an effective amount for in vivo imaging or where thepharmaceutical compositions are tested in an in vitro assay.

V. COMPOSITIONS

Each of the antibodies of the present invention can be used as acomposition when combined with a pharmaceutically acceptable carrier orexcipient. Such pharmaceutical compositions are useful foradministration to a subject in vivo or ex vivo, and for diagnosingand/or treating a subject with the disclosed antibodies, for example.

Pharmaceutically acceptable carriers are physiologically acceptable tothe administered patient and retain the therapeutic properties of theantibodies or peptides with which it is administered.Pharmaceutically-acceptable carriers and their formulations are andgenerally described in, for example, Remington′ pharmaceutical Sciences(18^(th) Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa.1990). One exemplary pharmaceutical carrier is physiological saline. Thephrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antibodies orpeptides from the administration site of one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Nor should apharmaceutically acceptable carrier alter the specific activity of theantagonists. Exemplary carriers and excipients have been providedelsewhere herein.

In one aspect, the present invention provides pharmaceuticallyacceptable or physiologically acceptable compositions including solvents(aqueous or non-aqueous), solutions, emulsions, dispersion media,coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration. Pharmaceuticalcompositions or pharmaceutical formulations therefore refer to acomposition suitable for pharmaceutical use in a subject. Thepharmaceutical compositions and formulations include an amount of aninvention compound, for example, an effective amount of an antagonist ofthe invention, and a pharmaceutically or physiologically acceptablecarrier.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration, systemic or local. Thus,pharmaceutical compositions include carriers, diluents, or excipientssuitable for administration by various routes.

In a further invention, the compositions of the present inventionfurther comprise a pharmaceutically acceptable additive in order toimprove the stability of the antagonist in composition and/or to controlthe release rate of the composition. Pharmaceutically acceptableadditives of the present invention do not alter the specific activity ofthe subject antagonist. A preferable pharmaceutically acceptableadditive is a sugar such as mannitol, sorbitol, glucose, xylitol,trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose,lactose and mixtures thereof. Pharmaceutically acceptable additives ofthe present invention can be combined with pharmaceutically acceptablecarriers and/or excipients such as dextrose. Alternatively, a preferablepharmaceutically acceptable additive is a surfactant such as polysorbate20 or polysorbate 80 to increase stability of the peptide and decreasegelling of the pharmaceutical solution. The surfactant can be added tothe composition in an amount of 0.01% to 5% of the solution. Addition ofsuch pharmaceutically acceptable additives increases the stability andhalf-life of the composition in storage.

The formulation and delivery methods will generally be adapted accordingto the site and the disease to be treated. Exemplary formulationsinclude, but are not limited to, those suitable for parenteraladministration, e.g., intravenous, intra-arterial, intramuscular, orsubcutaneous administration, including formulations encapsulated inmicelles, liposomes or drug-release capsules (active agents incorporatedwithin a biocompatible coating designed for slow-release); ingestibleformulations; formulations for topical use, such as creams, ointmentsand gels; and other formulations such as inhalants, aerosols and sprays.The dosage of the compounds of the invention will vary according to theextent and severity of the need for treatment, the activity of theadministered composition, the general health of the subject, and otherconsiderations well known to the skilled artisan.

Formulations or enteral (oral) administration can be contained in atablet (coated or uncoated), capsule (hard or soft), microsphere,emulsion, powder, granule, crystal, suspension, syrup or elixir.Conventional nontoxic solid carriers which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, can be used to prepare solid formulations. Supplementaryactive compounds (e.g., preservatives, antibacterial, antiviral andantifungal agents) can also be incorporated into the formulations. Aliquid formulation can also be used for enteral administration. Thecarrier can be selected from various oils including petroleum, animal,vegetable or synthetic, for example, peanut oil, soybean oil, mineraloil, sesame oil. Suitable pharmaceutical excipients include e.g.,starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol.

Pharmaceutical compositions for enteral, parenteral, or transmucosaldelivery include, for example, water, saline, phosphate buffered saline,Hank's solution, Ringer's solution, dextrose/saline, and glucosesolutions. The formulations can contain auxiliary substances toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents, detergents and the like. Additives canalso include additional active ingredients such as bactericidal agents,or stabilizers. For example, the solution can contain sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate or triethanolamine oleate. Additional parenteralformulations and methods are described in Bai (1997) J. Neuroimmunol.80:65 75; Warren (1997) J. Neurol. Sci. 152:31 38; and Tonegawa (1997)J. Exp. Med. 186:507 515. The parenteral preparation can be enclosed inampules, disposable syringes or multiple dose vials made of glass orplastic.

Pharmaceutical compositions for intradermal or subcutaneousadministration can include a sterile diluent, such as water, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid, glutathione orsodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

Pharmaceutical compositions for injection include aqueous solutions(where water soluble) or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, and sodium chloride may beincluded in the composition. The resulting solutions can be packaged foruse as is, or lyophilized; the lyophilized preparation can later becombined with a sterile solution prior to administration.

Pharmaceutically acceptable carriers can contain a compound thatstabilizes, increases or delays absorption or clearance. Such compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans; low molecular weight proteins; compositions that reduce theclearance or hydrolysis of peptides; or excipients or other stabilizersand/or buffers. Agents that delay absorption include, for example,aluminum monostearate and gelatin. Detergents can also be used tostabilize or to increase or decrease the absorption of thepharmaceutical composition, including liposomal carriers. To protectfrom digestion the compound can be complexed with a composition torender it resistant to acidic and enzymatic hydrolysis, or the compoundcan be complexed in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are known in theart (see, e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J.Pharm. Pharmacol. 48:119 135; and U.S. Pat. No. 5,391,377, describinglipid compositions for oral delivery of therapeutic agents).

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be through nasal sprays orsuppositories (see, e.g., Sayani (1996) “Systemic delivery of peptidesand proteins across absorptive mucosae” Crit. Rev. Ther. Drug CarrierSyst. 13:85 184). For transdermal administration, the active compoundcan be formulated into ointments, salves, gels, or creams as generallyknown in the art. Transdermal delivery systems can also be achievedusing patches.

For inhalation delivery, the pharmaceutical formulation can beadministered in the form of an aerosol or mist. For aerosoladministration, the formulation can be supplied in finely divided formalong with a surfactant and propellant. In another embodiment, thedevice for delivering the formulation to respiratory tissue is in whichthe formulation vaporizes. Other delivery systems known in the artinclude dry powder aerosols, liquid delivery systems, inhalers, air jetnebulizers and propellant systems (see, e.g., Patton (1998)Biotechniques 16:141 143; Dura Pharmaceuticals, San Diego, Calif.;Aradigm, Hayward, Calif.; Aerogen, Santa Clara, Calif.; and InhaleTherapeutic Systems, San Carlos, Calif.).

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations are known to those skilled in the art. The materials canalso be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to cells or tissues using antibodies or viral coat proteins)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known in the art, for example, asdescribed in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,522,811; 4,837,028;6,110,490; 6,096,716; 5,283,185; 5,279,833; Akimaru (1995) CytokinesMol. Ther. 1:197 210; Alving (1995) Immunol. Rev. 145: 5 31; and Szoka(1980) Ann Rev. Biophys. Bioeng. 9:467). Biodegradable microspheres orcapsules or other biodegradable polymer configurations capable ofsustained delivery of small molecules including peptides are known inthe art (see, e.g., Putney (1998) Nat. Biotechnol. 16:153 157).Compounds of the invention can be incorporated within micelles (see,e.g., Suntres (1994) J. Pharm. Pharmacol. 46:23 28; Woodle (1992) Pharm.Res. 9:260 265). Antagonists can be attached to the surface of the lipidmonolayer or bilayer. For example, antagonists can be attached tohydrazide-PEG-(distearoylphosphatidy-1) ethanolamine-containingliposomes (see, e.g., Zalipsky (1995) Bioconjug. Chem. 6: 705 708).Alternatively, any form of lipid membrane, such as a planar lipidmembrane or the cell membrane of an intact cell, e.g., a red blood cell,can be used. Liposomal and lipid-containing formulations can bedelivered by any means, including, for example, intravenous, transdermal(see, e.g., Vutla (1996) J. Pharm. Sci. 85:5 8), transmucosal, or oraladministration.

Compositions of the present invention can be combined with othertherapeutic moieties or imaging/diagnostic moieties as provided herein.Therapeutic moieties and/or imaging moieties can be provided as aseparate composition, or as a conjugated moiety. Linkers can be includedfor conjugated moieties as needed and have been described elsewhereherein.

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286 288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

Lipofections or liposomes can also be used to deliver the anti-LOXantibody, or an antibody fragment, into cells. Where antibody fragmentsare used, the smallest inhibitory fragment that specifically binds tothe binding domain of the target protein can be used. For example, basedupon the variable-region sequences of an antibody, peptide molecules canbe designed that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889 7893 (1993). The formulation herein can also containmore than one active compound as necessary for the particular indicationbeing treated, including, for example, those with complementaryactivities that do not adversely affect each other. Alternatively, or inaddition, the composition can comprise an agent that enhances itsfunction, such as, for example, a cytotoxic agent, cytokine,chemotherapeutic agent, or growth-inhibitory agent. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended. The active ingredients can also be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

Formulations for in vivo administration are sterile. Sterilization canbe readily accomplished via filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT®(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Various other pharmaceutical compositions and techniques for theirpreparation and use will be known to those of skill in the art in lightof the present disclosure. For a detailed listing of suitablepharmacological compositions and associated administrative techniquesone can refer to the detailed teachings herein, which can be furthersupplemented by texts such as Remington: The Science and Practice ofPharmacy 20th Ed. (Lippincott, Williams & Wilkins 2003).

Pharmaceutical compositions contemplated by the present invention havebeen described above. In one embodiment of the present invention, thepharmaceutical compositions are formulated to be free of pyrogens suchthat they are acceptable for administration to human patients. Testingpharmaceutical compositions for pyrogens and preparing pharmaceuticalcompositions free of pyrogens are well understood to one of ordinaryskill in the art.

One embodiment of the present invention contemplates the use of any ofthe pharmaceutical compositions of the present invention to make amedicament for treating a disorder of the present invention. Medicamentscan be formulated based on the physical characteristics of thepatient/subject needing treatment, and can be formulated in single ormultiple formulations based on the stage of the cancerous tissue.Medicaments of the present invention can be packaged in a suitablepharmaceutical package with appropriate labels for the distribution tohospitals and clinics wherein the label is for the indication oftreating a disorder as described herein in a subject. Medicaments can bepackaged as a single or multiple units. Instructions for the dosage andadministration of the pharmaceutical compositions of the presentinvention can be included with the pharmaceutical packages and kitsdescribed below.

VI. AFFINITY PURIFICATION

Anti-LOX antibodies and anti-LOXL2 antibodies described herein areuseful for affinity purification of LOX of LOXL2 from recombinant cellculture, natural sources or tissue biopsy samples (tissue and/or serum).In this process, antibodies against LOX or LOXL2 are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the LOX or LOXL2 to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the LOX or LOXL2, which is boundto the immobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the LOX or LOXL2 from the antibody.

VII. PACKAGES AND KITS

One embodiment of the present application includes a pharmaceuticalpackage or kit useful for the methods provided herein. One embodiment ofsuch pharmaceutical packages or kits includes preparations(compositions) of the antagonists as provided herein.

One aspect of the present invention relates to kits for carrying out theadministration of a LOX/LOXL2 inhibitor. Another aspect of the presentinvention relates to kits for carrying out the combined administrationof the LOX/LOXL2 inhibitor with one or more other therapeutic agent. Inone embodiment, the kit comprises a LOX/LOXL2 inhibitor formulated in apharmaceutical carrier or excipient, and at least one therapeutic agentthat is not said LOX/LOXL2 inhibitor, formulated as appropriate, in oneor more separate pharmaceutical preparations.

Pharmaceutical packages and kits can additionally include an excipient,a carrier, a buffering agent, a preservative or a stabilizing agent in apharmaceutical formulation. Each component of the kit can be enclosedwithin an individual container and all of the various containers can bewithin a single package. Invention kits can be designed for roomtemperature or cold storage.

Additionally, the preparations can contain stabilizers to increase theshelf-life of the kits and include, for example, bovine serum albumin(BSA) or other known conventional stabilizers. Where the compositionsare lyophilized, the kit can contain further preparations of solutionsto reconstitute the preparations. Acceptable solutions are well known inthe art and include, for example, pharmaceutically acceptable phosphatebuffered saline (PBS).

Additionally, the pharmaceutical packages or kits provided herein canfurther include any of the other moieties provided herein such as, forexample, a chemotherapeutic agent as described elsewhere in more detail.

Pharmaceutical packages and kits of the present invention can furtherinclude the components for an assay provided herein, such as, forexample, an ELISA assay. Alternatively, preparations of the kits areused in immunoassays, such as immunohistochemistry to test patienttissue biopsy sections. Pharmaceutical packages and kits of the presentinvention can further include the components for collection of a sample.

Pharmaceutical packages and kits of the present invention can furtherinclude a label specifying, for example, a product description, mode ofadministration and indication of treatment. Pharmaceutical packagesprovided herein can include any of the compositions as described herein.The pharmaceutical package can further include a label for preventing,reducing the risk of, or treating any of the disease indicationsdescribed herein.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions. Kits of the invention therefore can additionally includelabels or instructions for using the kit components in any method of theinvention. A kit can include an invention compound in a pack, ordispenser together with instructions for administering the compound in amethod of the invention.

Instructions can include instructions for practicing any of the methodsof the invention described herein including treatment, detection,monitoring or diagnostic methods. Instructions may additionally includeindications of a satisfactory clinical endpoint or any adverse symptomsthat may occur, or additional information required by regulatoryagencies such as the Food and Drug Administration for use on a humansubject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin or affixed to the kit, or on a label affixed to the kit orpackaging material, or attached to a vial or tube containing a componentof the kit. Instructions may additionally be included on a computerreadable medium, such as a disk (floppy diskette or hard disk), opticalCD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage mediasuch as RAM and ROM, IC tip and hybrids of these such asmagnetic/optical storage media.

The compositions of the kit of the present invention can be formulatedin single or multiple units for either a single test or multiple tests.

In preferred embodiments, the preparations of the kit are free ofpyrogens. Methods for testing for the presence of, and/or specificlevels of, pyrogens are routine in the art and kits are commerciallyavailable for such purpose.

Provided herein is a kit for treating a condition associated with LOX orLOXL2, containing a composition of an antibody or antigen bindingfragment thereof described herein and a pharmaceutically acceptablecarrier or excipient. A condition associated with LOX or LOXL2 can be,for example, a tumor, a metastasis, angiogenesis, or fibrosis. In oneembodiment, antibodies in such kits can comprise a detectable label, atherapeutic label or both. In another embodiment, antibodies in suchkits can be lyophilized.

Another aspect of the present invention relates to kits for carrying outthe combined administration of the LOX or LOXL2 inhibitor with othertherapeutic compounds. In one embodiment, the kit comprises a LOX orLOXL2 inhibitor formulated in a pharmaceutical carrier, and at least onecytotoxic agent, formulated as appropriate, in one or more separatepharmaceutical preparations.

VIII. DIAGNOSTIC METHODS

The present invention also provides methods for diagnosing, monitoring,staging or detecting the diseases described above by using agents thatrecognize different forms of LOX or LOXL2. For example, as describedabove, antibodies against different forms of LOX or LOXL2, thepreproprotein, secreted, mature or active form, can be used for thesepurposes. Methods of diagnosing, monitoring, staging or detecting thediseases described above by using agents that recognize different formsof LOX or LOXL2 are intended to encompass all of the diseases andindications described herein.

As described above, active LOX or LOXL2 is cleaved and can be detectedby virtue of its change in molecular weight (immunoblot) or by use ofantibodies that detect the uncleaved vs. cleaved form of LOX/LOXL, alongwith cellular localization by using various detection methods such asimmunohistochemistry (IHC).

It is believed that the extracellular matrix and conditioned mediumshould contain proteolytically processed, active LOX or LOXL whereasuncleaved, inactive LOX/LOXL should be localized intracellularly. Someactive, cleaved LOX/LOXL can also be detected inside the cell as aconsequence of uptake from the extracellular space.

Samples from individuals can be collected and analyzed by determininginactive or active LOX levels. This analysis can be performed prior tothe initiation of treatment using lysyl oxidase-specific therapy toidentify tumors having elevated active LOX/LOXL expression or activity.Such diagnosis analysis can be performed using any sample, including butnot limited to cells, protein or membrane extracts of cells, biologicalfluids such as sputum, blood, serum, plasma, or urine, or biologicalsamples such as tissue samples, formalin-fixed or frozen tissuesections.

Any suitable method for detection and analysis of inactive and/or activeLOX/LOXL can be employed. As used herein, the term “sample” refers to asample from a human, animal, or to a research sample, e.g., a cell,tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulatedmaterial. Samples also include, but are not limited to, protein ormembrane extracts of cells, biological fluids such as sputum, blood,serum, plasma, or urine, or biological samples such as formalin-fixed orfrozen tissue sections employing antibodies described herein. The term“sample” can also refer to a cell, tissue, organ, or fluid that isfreshly taken from a human or animal, or to a cell, tissue, organ, orfluid that is processed or stored. The sample can be tested in vivo,e.g., without removal from the human or animal, or it can be tested invitro. The sample can be tested after processing, e.g., by histologicalmethods.

Various diagnostic assay techniques known in the art can be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety directly or indirectly produces a detectable signal.For example, the detectable moiety can be any of those described hereinsuch as, for example, a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate (FITC), Texas red, cyanin, photocyan, rhodamine, orluciferin, or an enzyme, such as alkaline phosphatase, β-galactosidaseor horseradish peroxidase. Any method known in the art for conjugatingthe antibody to the detectable moiety can be employed, including thosemethods described by Hunter et al., Nature, 144:945 (1962); David etal., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).

Provided herein is a method of diagnosing a condition associated withLOX or LOXL2 comprising assessing a level of LOX and/or LOXL2 in asample of a subject, wherein a change in level of LOX and/or LOXL2 inthe sample in comparison with a reference sample indicates the presenceor increase of a tumor or metastasis. In one aspect, the conditionassociated with LOX or LOXL2 is a tumor, a metastasis, angiogenesis, orfibrosis. An increase in LOX and/or LOXL2 levels in the sample incomparison with a reference sample can indicate the presence of a tumoror metastasis or an increase in tumor or metastatic growth. Thereference sample can be a sample taken from the subject at an earliertime point or a sample from another individual. The level of LOX and/orLOXL2 levels in the sample can be detected by contacting the sample withan antibody or antigen binding fragment thereof described herein. In oneembodiment, the antibody or antigen binding fragment thereof isdetectably labeled.

In one embodiment, a method is provided for diagnosing cancer metastasisin a subject, comprising: assessing active LOX or LOXL2 levels oractivity in the blood, whereby a change in active LOX or LOXL2 levels oractivity (e.g., in gene expression, enzymatic activity, etc.) in theblood in comparison with a reference sample, indicates the presence ofmetastatic tumor growth. In some instances, the active LOX or LOXL2levels or activities in the blood can be lower than those when measuredearlier, which can indicate that the subject is at a greater risk ofcancer metastasis; that the cancer has metastasized; or that cancermetastasis has increased. The reference sample may derive from the samesubject, taken from the same tumor at a different time point or fromother site of the body, or from another individual.

In another embodiment, a method is provided for diagnosing cancermetastasis in a subject having a tumor, comprising: assessing active LOXor LOXL2 levels or activity in the tumor, whereby a change in active LOXor LOXL2 levels or activity in the tumor in comparison with a referencesample indicates the presence of metastatic tumor growth. In someinstances, the active LOX or LOXL2 levels or activities in the tumor canbe higher than those when measured earlier, which can indicate that thesubject is at a greater risk of cancer metastasis; that the cancer hasmetastasized; or that cancer metastasis has increased. The referencesample can derive from the same subject, taken from the same tumor at adifferent time point or from other site of the body, or from anotherindividual.

Also provided herein is a method for staging tumor growth or metastasisin a subject, comprising assessing LOX and/or LOXL2 (e.g., hLOX orhLOXL2) levels in a tumor of the subject, whereby a change in LOX and/orLOXL2 level (e.g., in gene expression or enzymatic activity) in thetumor in comparison with a reference sample, indicates the presence ofmetastatic tumor growth. In some instances, the LOX and/or LOXL2 levelsor activities in the tumor may be higher than those when measuredearlier for the same subject, or higher than those in a reference sampletaken from a normal tissue, which may indicate that the patient is at agreater risk of tumor metastasis; that the tumor has metastasized; orthat tumor metastasis has increased.

Staging of solid tumor cancers is well known. The TNM system is one ofthe most commonly used staging systems. This system has been accepted bythe International Union Against Cancer (UICC) and the American JointCommittee on Cancer (AJCC). Most medical facilities use the TNM systemas their main method for cancer reporting. PDQ®, the NCI's comprehensivecancer database, also uses the TNM system. The TNM system, referred toherein as “staging,” is based on the extent of the tumor, the extent ofspread to the lymph nodes, and the presence of metastasis.

Also provided here is a method for monitoring a subject's response to atherapy including a modulator of LOX/LOXL2 such as the treatment ofcancer, tumors, and fibrotic diseases. The method comprises: detecting achange in the level of C-reactive protein in the subject afteradministration of a modulator of LOX or LOXL2 to the subject, whereinthe change indicates that the LOX or LOXL2 modulator has a therapeuticeffect on the subject. A C-reactive protein is an importantpharmacodynamic marker for systemic inflammation. It is believed that areduced level of C-reactive protein (e.g., in the blood sample of thesubject) as compared to that prior to the administration of the LOX orLOXL2 inhibitor is indicative of the subject's response to the therapyusing an inhibitor of LOX or LOXL2.

Measurement of active LOX or LOXL2 levels can take the form of animmunological assay, which detects the presence of an active LOX orLOXL2 protein with an antibody to the protein, preferably an antibodyspecifically binding to active LOX or LOXL2. Antigen-binding fragmentsof anti-LOX/LOXL antibodies can also be used.

Immunoassays also can be used in conjunction with laser inducedfluorescence (see, for example, Schmalzing and Nashabeh, Electrophoresis18:2184-93 (1997); and Bao, J. Chromatogr. B. Biomed. Sci. 699:463-80(1997), each of which is incorporated herein by reference). Liposomeimmunoassays, such as flow-injection liposome immunoassays and liposomeimmunosensors also can be used to determine active LOX or LOXL levelsaccording to a method of the invention (Rongen et al., J. Immunol.Methods 204:105-133 (1997). Immunoassays, such as enzyme-linkedimmunosorbent assays (ELISAs), can be particularly useful in a method ofthe invention. A radioimmunoassay also can be useful for determiningwhether a sample is positive for active LOX or LOXL2 or for determiningthe level of active LOX or LOXL2. A radioimmunoassay using, for example,an iodine-125 labeled secondary antibody, can be used.

In addition, one can measure the activity of active LOX or LOXL2, thusignoring the amount of inactive enzyme. Enzymatic activity of active LOXor LOXL2 can be measured in a number of ways, using a soluble elastin orsoluble collagen with labeled lysine as a substrate. Details of anactivity assay are given in Royce et al., “Copper metabolism in mottledmouse mutants. The effect of copper therapy on lysyl oxidase activity inbrindled (Mobr) mice,” Biochem J. 1982 Feb. 15; 202(2): 369-371. Anexemplary assay is a chromogenic assay, such as that described byPalamakumbura, et al. “A fluorometric assay for detection of lysyloxidase enzyme activity in biological samples,” Anal Biochem. 2002 Jan.15; 300(2):245-51.

In addition to measuring the level of LOX or LOXL2 in the blood (orurine), one can measure secondary products of LOX or LOXL2 activity. Forexample, deoxypyridinoline (Dpd) is formed by the enzymatic action oflysyl oxidase on lysine residues. Dpd is released into the circulationas a result of osteoclastic degradation of bone. It cannot be re-used,is cleared by the kidney and is excreted unchanged in urine. Thus, atest based on the Immunodiagnostic Systems (IDS) Gamma BCT Dpd assay,using a coated tube RIA using an anti-Dpd monoclonal antibody can beused to measure enzymatic activity.

Anti-LOX antibodies and anti-LOXL2 antibodies described herein can alsobe used in the diagnosis of diseases or conditions associated withaberrant collagen metabolism such as various fibrotic conditions, forexample, lung fibrosis, as well as in proliferative vitreousretinopathy, surgical scarring, systemic sclerosis, scleroderma, woundcontraction, hypertrophic scars, fibromatosis (especially Dupuytren'sdisease), and keloids.

IX. THERAPEUTIC METHODS

The pharmaceutical formulations according to the present invention canbe used to treat a wide variety of diseases and disorders such as, forexample, cancer, metastasis, fibrosis and aberrant angiogenesis.

Provided herein is a method of inhibiting LOXL2 by contacting a sampleor a cellular tissue with any of the anti-LOXL2 antibodies or antigenbinding fragments thereof described herein. Binding of said antibody orantigen binding fragment thereof to LOXL2 inhibits enzymatic activity ofLOXL2.

Also provided herein is a method of inhibiting LOX by contacting asample or cellular tissue with any of the anti-LOX antibodies or antigenbinding fragments thereof described herein. Binding of said antibody orantigen binding fragment thereof to LOX inhibits enzymatic activity ofLOX.

In either of such methods, contacting can occur in vitro, in vivo or exvivo.

Inhibition of LOX or LOXL2 can have one or more effects in a subjectsuch as, for example, reduction in tumor growth, reduction inangiogenesis, reduction in a fibrotic disease, and/or decreasingextracellular matrix formation. Fibrotic diseases include, but are notlimited to liver fibrosis, lung fibrosis, kidney fibrosis, cardiacfibrosis, and scleroderma.

Provided herein are methods of treating diseases and disordersassociated with aberrant expression of LOX or LOXL2. Diseases anddisorder include, but are not limited to tumors (e.g., primary ormetastatic), angiogenesis related conditions and fibrotic conditions.

As used herein, “prevention” refers to prophylaxis, prevention of onsetof symptoms, prevention of progression of a disease or disorderassociated with fibrosis or correlated with LOX/LOXL2 activity. As usedherein, “treat” or “treatment” means stasis or a postponement ofdevelopment of the symptoms associated a disease or disorder describedherein. The terms further include ameliorating existing uncontrolled orunwanted symptoms, preventing additional symptoms, and ameliorating orpreventing the underlying metabolic causes of symptoms. Thus, the termsdenote that a beneficial result has been conferred on a mammaliansubject with a disease or symptom, or with the potential to develop suchdisease or symptom. A response is achieved when the patient experiencespartial or total alleviation, or reduction of signs or symptoms ofillness, and specifically includes, without limitation, prolongation ofsurvival. The expected progression-free survival times can be measuredin months to years, depending on prognostic factors including the numberof relapses, stage of disease, and other factors. Prolonging survivalincludes without limitation times of at least 1 month (mo), about atleast 2 months (mos.), about at least 3 mos., about at least 4 mos.,about at least 6 mos., about at least 1 year, about at least 2 years,about at least 3 years, or more. Overall survival can also be measuredin months to years. The patient's symptoms can remain static or candecrease.

Pharmaceutical compositions of the present invention are administered intherapeutically effective amounts which are effective for producing somedesired therapeutic effect at a reasonable benefit/risk ratio applicableto any medical treatment. For the administration of the presentpharmaceutical compositions to human patients, the pharmaceuticalcompositions of the present invention can be formulated by methodologyknown by one of ordinary skill in the art to be substantially free ofpyrogens such that they do not induce an inflammatory response.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of a therapeutic agent that whenadministered alone or in combination with another therapeutic agent to acell, tissue, or subject is effective to prevent or ameliorate thedisease condition or the progression of the disease. A therapeuticallyeffective dose further refers to that amount of the compound sufficientto result in amelioration of symptoms, e.g., treatment, healing,prevention or amelioration of the relevant medical condition, or anincrease in rate of treatment, healing, prevention or amelioration ofsuch conditions. When applied to an individual active ingredientadministered alone, a therapeutically effective dose refers to thatingredient alone. When applied to a combination, a therapeuticallyeffective dose refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously. For example, when in vivo administration ofan anti-LOX/anti-LOXL2 antibody is employed, normal dosage amounts canvary from about 10 ng/kg to up to 100 mg/kg of mammal body weight ormore per day, preferably about 1 μg/kg/day to 50 mg/kg/day, optionallyabout 100 μg/kg/day to 20 mg/kg/day, 500 μg/kg/day to 10 mg/kg/day, or 1mg/kg/day to 10 mg/kg/day, depending upon the route of administration.

An effective response of the present invention is achieved when thepatient experiences partial or total alleviation or reduction of signsor symptoms of illness, and specifically includes, without limitation,prolongation of survival. The expected progression-free survival timesmay be measured in months to years, depending on prognostic factorsincluding the number of relapses, stage of disease, and other factors.Prolonging survival includes without limitation times of at least 1month (mo), about at least 2 mos., about at least 3 mos., about at least4 mos., about at least 6 mos., about at least 1 year, about at least 2years, about at least 3 years, or more. Overall survival is alsomeasured in months to years. The patient's symptoms may remain static,and the tumor burden may not increase.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount (ED50) of thepharmaceutical composition required. For example, the physician orveterinarian can start doses of the compounds of the invention employedin the pharmaceutical composition at levels lower than that required inorder to achieve the desired therapeutic effect and gradually increasethe dosage until the desired effect is achieved.

As used herein, the term “subject” means mammalian subjects. Exemplarysubjects include, but are not limited to humans, monkeys, dogs, cats,mice, rats, cows, horses, goats and sheep. In some embodiments, thesubject has cancer and can be treated with the agent of the presentinvention as described below.

Regardless of the route of administration selected, the compounds of thepresent invention, which are used in a suitably hydrated form, and/orthe pharmaceutical compositions of the present invention are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular composition employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

In one aspect provided herein, administration of the antibodies resultsin an improvement the subject's condition. In another aspect,administration of the antibodies prevents the subject's condition fromworsening and/or prolongs survival of the patient.

The patient can be a mammal such as a human or a non-human. Such apatient can be symptomatic or asymptomatic.

Compositions can be administered locally, regionally or systemically byany suitable route provided herein.

In one aspect, symptoms of the patient are ameliorated. Amelioration canbe manifested as, for example, reduction in pain, reduced tumor size,elimination of tumors, prevention of increases in tumor size orprogression or of disease, prevention of formation of metastasis, orinhibition of metastatic growth, inhibition of fibrosis, inhibition ofangiogenesis, or a combination thereof.

If needed, for cancer treatments, methods can further include surgicalremoval of the cancer and/or administration of an anti-cancer agent ortreatment. Administration of such an anti-cancer agent or treatment canbe concurrent with administration of the compositions disclosed herein.Anti-cancer agents have been provided elsewhere herein.

In one aspect, administration of any of the antibodies provided hereinreduces or eliminates the need for the patient to undergo surgery ortreatment with one or more anti-cancer agents or treatments.

Indications that can be treated using the pharmaceutical formulations ofthe present invention include those involving undesirable oruncontrolled cell proliferation. Such indications include benign tumors,various types of cancers such as primary tumors and tumor metastasis,restenosis (e.g., coronary, carotid, and cerebral lesions),hematological disorders, abnormal stimulation of endothelial cells(atherosclerosis), insults to body tissue due to surgery, abnormal woundhealing, abnormal angiogenesis, diseases that produce fibrosis oftissue, macular degeneration, glaucoma; age-related macular degeneration(wet AMD and dry AMD), atherosclerosis, rheumatoid arthritis, multiplesclerosis, liver fibrosis, kidney fibrosis, lung fibrosis, scleroderma,atherosclerosis, and Alzheimer's disease, repetitive motion disorders,disorders of tissues that are not highly vascularized, and proliferativeresponses associated with organ transplants.

Liver fibrosis includes, but is not limited to, cirrhosis, andassociated conditions such as chronic viral hepatitis, non-alcoholicfatty liver disease (NAFLD), alcoholic steatohepatitis (ASH),non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC),biliary cirrhosis, and autoimmune hepatitis.

Lung fibrosis includes, but is not limited to, idiopathic pulmonaryfibrosis (IPF) or cryptogenic fibrosing alveolitis, chronic fibrosinginterstitial pneumonia, interstitial lung disease (ILD), diffuseparenchymal lung disease (DPLD), emphysema and chronic obstructivepulmonary disease (COPD), and chronic asthma.

Cardiac fibrosis includes, but is not limited to, congestive heartfailure, cardiomyopathy, and post-myocardial infarction defects in heartfunction.

Kidney fibrosis includes, but is not limited to, diabetic nephropathy,vesicoureteral reflux, tubulointerstitial renal fibrosis;glomerulonephritis or glomerular nephritis, including focal segmentalglomerulosclerosis and membranous glomerulonephritis, andMesangiocapillary glomerular nephritis.

Generally, cells in a benign tumor retain their differentiated featuresand do not divide in a completely uncontrolled manner. A benign tumor isusually localized and non-metastatic. Specific types benign tumors thatcan be treated using the present invention include hemangiomas,hepatocellular adenoma, cavernous haemangioma, focal nodularhyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bileduct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas, pyogenicgranulomas, moles, uterine fibroids, thyroid adenomas, adrenocorticaladenomas, and pituitary adenomas.

In a malignant tumor cells become undifferentiated, do not respond tothe body's growth control signals, and multiply in an uncontrolledmanner. The malignant tumor is invasive and capable of spreading todistant sites (metastasizing). Malignant tumors are generally dividedinto two categories: primary and secondary. Primary tumors arisedirectly from the tissue in which they are found. A secondary tumor, ormetastasis, is a tumor which is originated elsewhere in the body but hasnow spread to a distant organ. The common routes for metastasis aredirect growth into adjacent structures, spread through the vascular orlymphatic systems, and tracking along tissue planes and body spaces(peritoneal fluid, cerebrospinal fluid, etc.)

Specific types of cancers or malignant tumors, either primary orsecondary, that can be treated using this invention include, but are notlimited to, lung cancer (including lung adenocarcinoma, squamous cellcarcinoma, large cell carcinoma, bronchioloalveolar carcinoma,non-small-cell carcinoma, small cell carcinoma, mesothelioma); breastcancer (including ductal carcinoma, lobular carcinoma, inflammatorybreast cancer, clear cell carcinoma, mucinous carcinoma,); colorectalcancer (colon cancer, rectal cancer); anal cancer; pancreatic cancer(including pancreatic adenocarcinoma, islet cell carcinoma,neuroendocrine tumors); prostate cancer; ovarian carcinoma (ovarianepithelial carcinoma or surface epithelial-stromal tumour includingserous tumour, endometrioid tumor and mucinous cystadenocarcinoma,sex-cord-stromal tumor); liver and bile duct carcinoma (includinghepatocelluar carcinoma, cholangiocarcinoma, hemangioma); esophagealcarcinoma (including esophageal adenocarcinoma and squamous cellcarcinoma); non-Hodgkin's lymphoma; bladder carcinoma; carcinoma of theuterus (including endometrial adenocarcinoma, uterine papillary serouscarcinoma, uterine clear-cell carcinoma, uterine sarcomas andleiomyosarcomas, mixed mullerian tumors); glioma, glioblastoma,medullablastoma, and other tumors of the brain; kidney cancers(including renal cell carcinoma, clear cell carcinoma, Wilm's tumor);cancer of the head and neck (including squamous cell carcinomas); cancerof the stomach (stomach adenocarcinoma, gastrointestinal stromal tumor);multiple myeloma; testicular cancer; germ cell tumor; neuroendocrinetumor; cervical cancer; carcinoids of the gastrointestinal tract,breast, and other organs; signet ring cell carcinoma; mesenchymal tumorsincluding sarcomas, fibrosarcomas, haemangioma, angiomatosis,haemangiopericytoma, pseudoangiomatous stromal hyperplasia,myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumour,lipoma, angiolipoma, granular cell tumour, neurofibroma, schwannoma,angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma ora leiomysarcoma.

The term “metastasis” means the ability of tumor cells to invade hosttissues and metastasize to distant, often specific organ sites. As isknown, this is the salient feature of lethal tumor growths. Metastasisformation occurs via a complex series of unique interactions betweentumor cells and normal host tissues and cells. In the context of thepresent invention, lysyl oxidase activity is critical in the metastaticgrowth of tumors, i.e., the growth of metastases, particularly underhypoxic conditions. As hypoxic tumors are also the most aggressive andresistant to traditional chemotherapy, agents modulating lysyl oxidaseexpression and/or function provide a novel therapy against metastatictumors, particularly chemo-resistant tumors. “Metastasis” isdistinguished from invasion. As described in “Understanding CancerSeries: Cancer,” in the world wide web site:cancer.gov/cancertopics/understandingcancer/cancer; invasion refers tothe direct migration and penetration by cancer cells into neighboringtissues.

Hematologic disorders include abnormal growth of blood cells which canlead to dysplastic changes in blood cells and hematologic malignanciessuch as various leukemias. Examples of hematologic disorders include butare not limited to acute myeloid leukemia, acute promyelocytic leukemia,acute lymphoblastic leukemia, chronic myelogenous leukemia, themyelodysplastic syndromes, and sickle cell anemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchromosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subgroup ofAML. This subtype is characterized by promyelocytic blasts containingthe 15;17 chromosomal translocation. This translocation leads to thegeneration of the fusion transcript comprised of the retinoic acidreceptor and a sequence PML.

Acute lymphoblastic leukemia (ALL) is a heterogenerous disease withdistinct clinical features displayed by various subtypes. Reoccurringcytogenetic abnormalities have been demonstrated in ALL. The most commoncytogenetic abnormality is the 9;22 translocation. The resultantPhiladelphia chromosome represents poor prognosis of the patient.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferativedisorder of a pluripotent stem cell. CML is characterized by a specificchromosomal abnormality involving the translocation of chromosomes 9 and22, creating the Philadelphia chromosome. Ionizing radiation isassociated with the development of CML.

The myelodysplastic syndromes (MDS) are heterogeneous clonalhematopoietic stem cell disorders grouped together because of thepresence of dysplastic changes in one or more of the hematopoieticlineages including dysplastic changes in the myeloid, erythroid, andmegakaryocytic series. These changes result in cytopenias in one or moreof the three lineages. Patients afflicted with MDS typically developcomplications related to anemia, neutropenia (infections), orthrombocytopenia (bleeding). Generally, from about 10% to about 70% ofpatients with MDS develop acute leukemia.

Administration of LOX or LOX2 inhibitors has been found to reduce thesize of existing tumors, to prevent metastases, and to reduce the sizeof (or even eliminate) existing metastases (see, e.g., Molnar et al.(2003) Biochim Biophys. Acta. 1647:220-224).

Provided herein is a method of reducing tumor growth in a subject, byadministering any of the anti-LOX or anti-LOXL2 antibodies or antigenbinding fragments thereof described herein. In one embodiment, a tumoris a primary tumor. In another embodiment, a tumor is a metastatictumor. Metastatic tumor burden of a subject can stabilized byadministering antibodies as described herein. Tumor in the subject canbe reduced by at least 10%, 25%, 50%, 70%, 90%, 95%, or more as comparedto the tumor in the subject prior to treatment.

When an antibody or antigen binding fragment thereof specifically bindsto LOXL2, examples of tumors include, but are not limited to, a colontumor, an esophageal tumor, a breast tumor, a prostate tumor, a squamouscarcinoma or a spindle cell carcinoma.

When an antibody or antigen binding fragment thereof specifically bindsto LOX, examples of tumors include, but are not limited to, a breasttumor, a lung tumor, a kidney tumor, a uterine tumor, a liver tumor, ora head and neck tumor.

Provided herein is a method for preventing or reducing tumor growth,metastatic tumor growth, in a subject in vivo, comprising administeringto a subject in need thereof an effective amount of an inhibitor of LOXor LOXL2 activity; and optionally, a pharmaceutically acceptable carrieror excipient, thereby preventing or reducing tumor growth, for exampleby at least 25%, 50%, 75%, 90%, or 95%, in the subject treated. Adetailed description of suitable compositions for use in the presenttreatment methods is given above. Such methods are useful, for example,when the tumor is hypoxic. Hypoxic tumors can be readily identifiedusing routine methods in the art. See, e.g., U.S. Pat. No. 5,674,693.

Also, provided herein is a method of treating metastasis in a subjectwith cancer in vivo, comprising administering to a subject in needthereof an effective amount of a LOX or LOXL2 inhibitor, therebyinhibiting metastasis, for example, by at least 25%, 50%, 75%, 90%, or95%, in the subject treated. In one embodiment, inhibitors specificallyinhibit human LOX or human LOXL2. Antibodies to be used in these methodshave been described above. The antibody may be desirable to minimizecross-reactions with other members of the LOX or LOXL2 families and,thus, reduce the potential adverse side effects due to complications andnormal tissue toxicity.

Also provided herein is a method of increasing or enhancing the chancesof survival of a subject with metastatic tumor, comprising administeringto a subject in need thereof an effective amount of an anti-LOX antibodyor an anti-LOXL2 antibody, thereby increasing or enhancing the chancesof survival of the subject treated by a certain period of time, forexample, by at least 10 days, 1 month, 3 months, 6 months, 1 year, 1.5years, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years, or more.The increase in survival of a subject can be defined, for example, asthe increase in survival of a preclinical animal model of cancermetastases (e.g., a mouse with metastatic cancer), by a certain periodof time, for example, by at least 10 days, 1 month, 3 months, 6 months,or 1 year, or at least 2 times, 3 times, 4 times, 5 times, 8 times, or10 times, more than a control animal model (that has the same type ofmetastatic cancer) without the treatment with the inventive method.Alternatively, the increase in survival of a mammal can also be defined,for example, as the increase in survival of a patient with cancermetastases by a certain period of time, for example, by at least 10days, 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years,4 years, 5 years, 8 years, 10 years or more than a patient with the sametype of metastatic cancer but without the treatment with the inventivemethod. The control patient may be on a placebo or treated withsupportive standard care such as chemical therapy, biologics and/orradiation that do not include the inventive method as a part of thetherapy.

Also provided herein is a method of stabilizing metastatic tumor burdenof a subject, comprising administering to a subject in need thereof aneffective amount of an anti-LOX antibody or an anti-LOXL2 antibody,thereby stabilizing metastatic tumor burden of a subject for a certainperiod of time, for example, for at least 10 days, 1 month, 3 months, 6months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 8 years,10 years or more. Stabilization of the metastatic tumor burden of asubject can be defined as stabilization of metastatic tumor burden of apreclinical animal model with metastatic tumor burden (e.g., a mousewith metastatic tumor) for a certain period of time, for example, for atleast 10 days, 1 month, 3 months, 6 months, or 1 year more than acontrol animal model (that has the same type of metastatic tumor)without the treatment with the inventive method.

The present treatment methods also include a method to increase theefficacy of chemotherapeutic agents, comprising administering to asubject in need thereof an effective amount of an anti-LOX antibody oran anti-LOXL2 antibody; and optionally, a pharmaceutically acceptablecarrier or excipient, thereby increasing the efficacy ofchemotherapeutic agents (which are described in more detail above). Alsocontemplated are methods involving the delivery of LOX inhibitoryformulations in combination with radiation therapy. Radiation therapymay be used to treat almost every type of solid tumor, including cancersof the brain, breast, cervix, larynx, lung, pancreas, prostate, skin,spine, stomach, uterus, or soft tissue sarcomas. Radiation can also beused to treat leukemia and lymphoma (cancers of the blood-forming cellsand lymphatic system, respectively). Radiation dose to each site dependson a number of factors, including the type of cancer and whether thereare tissues and organs nearby that may be damaged by radiation.Radiation will typically be delivered as X-rays, where the dosage isdependent on the tissue being treated. Radiopharmaceuticals, also knownas radionucleotides, may also be used to treat cancer, including thyroidcancer, cancer that recurs in the chest wall, and pain caused by thespread of cancer to the bone (bone metastases). Radionuclides have beendescribed in more detail above.

The subject to be treated or diagnosed by the present methods includes asubject having or being at risk of having metastatic tumor growth. Suchtumors can be a In one aspect, a tumor is, for example, Lung cancer(including lung adenocarcinoma, squamous cell carcinoma, large cellcarcinoma, bronchioloalveolar carcinoma, non-small-cell carcinoma, smallcell carcinoma, mesothelioma); breast cancer (including ductalcarcinoma, lobular carcinoma, inflammatory breast cancer, clear cellcarcinoma, mucinous carcinoma,); colorectal cancer (colon cancer, rectalcancer); anal cancer; pancreatic cancer (including pancreaticadenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostatecancer; ovarian carcinoma (ovarian epithelial carcinoma or surfaceepithelial-stromal tumour including serous tumour, endometrioid tumorand mucinous cystadenocarcinoma, sex-cord-stromal tumor); liver and bileduct carcinoma (including hepatocelluar carcinoma, cholangiocarcinoma,hemangioma); esophageal carcinoma (including esophageal adenocarcinomaand squamous cell carcinoma); non-Hodgkin's lymphoma; bladder carcinoma;carcinoma of the uterus (including endometrial adenocarcinoma, uterinepapillary serous carcinoma, uterine clear-cell carcinoma, uterinesarcomas and leiomyosarcomas, mixed mullerian tumors); glioma,glioblastoma, medullablastoma, and other tumors of the brain; kidneycancers (including renal cell carcinoma, clear cell carcinoma, Wilm'stumor); cancer of the head and neck (including squamous cellcarcinomas); cancer of the stomach (stomach adenocarcinoma,gastrointestinal stromal tumor); multiple myeloma; testicular cancer;germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids ofthe gastrointestinal tract, breast, and other organs; signet ring cellcarcinoma; mesenchymal tumors including sarcomas, fibrosarcomas,haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatousstromal hyperplasia, myofibroblastoma, fibromatosis, inflammatorymyofibroblastic tumour, lipoma, angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma,osteosarcoma, leiomyoma or a leiomysarcoma. In one non-limitingembodiment, the tumor is a breast tumor, a pancreas tumor, a lung tumor,a cervical tumor, a colon tumor or a head and neck tumor.

The present invention also provides a method for preventing or reducingthe risk of tumor metastasis in a subject, comprising administering to asubject in need thereof an effective amount of an anti-LOX antibody oran anti-LOXL2 antibody; and optionally, a pharmaceutically acceptablecarrier or excipient, thereby preventing or reducing preventing orreducing the risk of tumor metastasis. The inhibitor can be an antibodyor an antigen binding fragment thereof. The subject in need of such aprophylactic can be an individual who is genetically predisposed tocancer or at a high risk of developing cancer due to various reasonssuch as family history of cancer and carcinogenic environment.

Examples of the human gene that is involved in the onset or developmentof cancer include, but are not limited to, VHL (the Von Hippon Landaugene involved in Renal Cell Carcinoma); P16/INK4A (involved inlymphoma); E-cadherin (involved in metastasis of breast, thyroid,gastric cancer); hMLH1 (involved in DNA repair in colon, gastric, andendometrial cancer); BRCA1 (involved in DNA repair in breast and ovariancancer); LKB1 (involved in colon and breast cancer); P15/INK4B (involvedin leukemia such as AML and ALL); ER (estrogen receptor, involved inbreast, colon cancer and leukemia); 06-MGMT (involved in DNA repair inbrain, colon, lung cancer and lymphoma); GST-pi (involved in breast,prostate, and renal cancer); TIMP-3 (tissue metalloprotease, involved incolon, renal, and brain cancer metastasis); DAPK1 (DAP kinase, involvedin apoptosis of B-cell lymphoma cells); P73 (involved in apoptosis oflymphomas cells); AR (androgen receptor, involved in prostate cancer);RAR-beta (retinoic acid receptor-beta, involved in prostate cancer);Endothelin-B receptor (involved in prostate cancer); Rb (involved incell cycle regulation of retinoblastoma); p53 (an important tumorsuppressor gene); P14ARF (involved in cell cycle regulation); RASSF1(involved in signal transduction); APC (involved in signaltransduction); Caspase-8 (involved in apoptosis); TERT (involved insenescence); TERC (involved in senescence); TMS-1 (involved inapoptosis); SOCS-1 (involved in growth factor response ofhepatocarcinoma); PITX2 (hepatocarcinoma breast cancer); MINT1; MINT2;GPR37; SDC4; MY0D1; MDR1; THBS1; PTC1; and pMDR1, as described inSantini et al. (2001) Ann of Intern. Med. 134:573-586, which is hereinincorporated by reference in its entirety. Nucleotide sequences of thesegenes can be retrieved from the website of the National Center forBiotechnology Information (NCBI).

It should be noted that, although leukemia is a cancer of the blood, itmight affect other organs, or, in effect, metastasize. In acuteleukemias, the abnormal cells may collect in the central nervous system,the testicles, the skin and any other organ in the body. Becauseleukemia already involves all of the bone marrow in the body, and inmany cases, has spread to other organs such as the liver, spleen, andlymph nodes, the staging of leukemia depends on other information thatreflects the patient's outlook for survival. Leukemias include, forexample, Acute lymphoblastic leukemia (ALL), Chronic lymphocyticleukemia (CLL), Acute myelogenous leukemia (AML), Chronic myelogenousleukemia (CML) and Hairy cell leukemia (HCL). Different staging systemsare used for different types of chronic leukemia. Some types do not haveany staging system. Methods of staging are described in more detailbelow.

Treatment of abnormal cell proliferation due to insults to body tissueduring surgery can be possible for a variety of surgical procedures,including joint surgery, bowel surgery, and keloid scarring. Diseasesthat produce fibrotic tissue include emphysema. Repetitive motiondisorders that can be treated using the present invention include carpaltunnel syndrome. An example of cell proliferative disorders that can betreated using the invention is a bone tumor. Provided herein is a methodfor preventing or reducing the risk of abnormal cell proliferation dueto insults to body tissue during surgery or a disease that producesfibrotic tissue in a subject, comprising administering to a subject inneed thereof an effective amount of an anti-LOX antibody or ananti-LOXL2 antibody; and optionally, a pharmaceutically acceptablecarrier or excipient, thereby preventing or reducing abnormal cellproliferation due to insults to body tissue during surgery. Theinhibitor can be an antibody or an antigen binding fragment thereof. Thesubject in need of such a prophylactic can be an individual who isgenetically predisposed to cancer or at a high risk of developing cancerdue to various reasons such as family history of cancer and carcinogenicenvironment. In one embodiment, the disease can be, for example, jointsurgery, bowel surgery, keloid scarring, a disease that producesfibrotic tissue, repetitive motion disorders of a bone tumor.

The proliferative responses associated with organ transplantation thatcan be treated using this invention include those proliferativeresponses contributing to potential organ rejections or associatedcomplications. Specifically, these proliferative responses can occurduring transplantation of the heart, lung, liver, kidney, and other bodyorgans or organ systems. Provided herein is a method of treating anabnormal proliferative response associated with organ transplantation,comprising administering to a subject in need thereof an effectiveamount of an anti-LOX antibody or an anti-LOXL2 antibody; andoptionally, a pharmaceutically acceptable carrier or excipient, therebypreventing or reducing abnormal cell proliferation due to organtransplantation. Transplantation can include, for example, transplant ofheart, lung, liver, kidney, and other body organs or organ systems.

The indication for the inventive composition also includes fibrosis.Fibrosis results from abnormal accumulation of fibrous tissue that canoccur as a part of the wound-healing process in damaged tissue. Suchtissue damage may result from physical injury, inflammation, infection,exposure to toxins, and other causes. Examples of fibrosis include liverfibrosis, lung fibrosis, kidney fibrosis, cardiac fibrosis andscleroderma. The compounds and agents of the described invention arealso contemplated for the treatment, prevention, and/or amelioration offibrotic conditions.

Fibrotic tissues accumulate in the heart and blood vessels as a resultof hypertension, hypertensive heart disease, atherosclerosis, andmyocardial infarction. High blood pressure, or hypertension, can because by a variety of factors and often leads to the development ofHypertensive Heart Disease (HHD) with progression to cardiac arrest andmyocardial infarction. Similarly, atherosclerosis and other ischemicheart diseases often also result in cardiac arrest. These cardiovasculardiseases all exhibit an accumulation of extra-cellular matrix orfibrotic deposition which results in stiffening of the vasculature andstiffening of the cardiac tissue itself. This deposition of fibroticmaterial is a response to the damage induced by the hypertensive and/orsclerotic state, but the effects of this response also result in thenegative effects of vascular and cardiac stiffening as well as ventricleenlargement. Additionally, it is believed that the increased cardiacfibrosis seen in cardiovascular disease disrupts or alters the signalstransmitted to cardiomyocytes via the tissue scaffolding of the heart,further leading to disruption of efficient cardiac function andpromoting cardiac arrest and myocardial infarction. Given the identifiedrole of increased extracellular matrix deposition in cardiac fibroses,the compounds of the present invention are useful for the prevention,treatment, and/or amelioration of cardiac fibroses by the inhibition ofLOX/LOXL2.

The prevent invention also provides compositions, methods, systems,medical devices or kits for the treatment or prevention of cardiacfibrosis associated with cardiovascular diseases such as hypertensiveheart disease (HHD), myocardial infarction (MI), atherosclerosis,restenosis (e.g. coronary, carotid, and cerebral lesions), and heartdisease associated with cardiac ischemic events.

The post MI-healing response can induce expression of LOX/LOXL2 but ifthis process continues unchecked, excessive cross-linking leads toextracellular matrix remodeling or fibrosis that results in cardiacdysfunction. The enzymes that break down matrices and cross-linkedcollagen or elastin appear to function more slowly or less efficientlyand are outpaced by the cross-linking events. As LOX/LOXL2 also plays arole in epithelial-mesenchymal transition (EMT), this contributesfurther to cardiomyocyte remodeling and cardiomyocyte hypertrophy, inaddition to matrix remodeling.

Initial reparative fibrosis induced by the MI may be helpful (e.g.,prevents aneurysm and related damage) and can be allowed to proceedunhindered. However, while not wishing to be bound to a particulartheory or mechanism of action, the inventors believe that anti-LOX/LOXL2treatment initiated following this reparative fibrosis phase couldattenuate reactive (mal-adaptive) fibrosis that leads to cardiacdysfunction. For example, anti-LOX/LOXL2 treatment can be initiated 2,4, 6, 8, 10, 12, 14, 16, 16, 20, 22, 24, 36, or 48 hours after MI,inclusive of all integers in-between. Additionally, anti-LOX/LOXL2treatment can be initiated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days after MI. Similarly, increases in blood pressure (hypertension)result in increased collagen deposition and reduced protein degradationin cardiac tissue. (Berk et al., J. Clin. Invest., 117(3): 568-575(2007)). Anti-LOX/LOXL2 treatment initiated following diagnosis and/orestablishment of Hypertensive Heart Disease or hypertension can prevent,reduce, or ameliorate fibrosis associated with hypertension. Suchanti-LOX/LOXL2 treatment is initiated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 days after increases in hypertension or systemic bloodpressure are diagnosed or detected.

As another example, biomarkers may be used to determine when aninappropriate level of cross-linking might be occurring: for example,LOX levels have been shown to correlate with C reactive protein (CRP), acommonly used biomarker, and treatment could begin when CRP levels areelevated above appropriate normal levels. More directly, methods andtest kits exist to measure the release of cross-linked collagentelopeptides in urine or blood. Elevated levels of these collagenfragments could indicate a transition from reparative fibrosis toreactive (mal-adaptive) fibrosis. In addition, measures of cardiacfunction and output, including those associated with efficientcontraction of the ventricle, can be made.

An inhibitor of LOX/LOXL2 can be delivered to a subject prior to,concurrently, or post a pathological cardiac condition or disease, suchas hypertension, hypertensive heart disease (HHD), myocardial infarction(MI), atherosclerosis, and restenosis, to prevent the onset of, toreduce the risk of, or to retreat pathological fibrosis associated withsuch a pathological cardiac condition or disease. For example, aninhibitor of LOX/LOXL2 can be administered at least 1 hr, 2 hrs, 3 hrs,5 hrs, or 10 hrs, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days after the onset of such a pathological cardiac condition ordisease.

Additionally, a limited duration of treatment is envisioned. Treatmentshould be sustained only long enough to prevent or attenuate reactivefibrosis to prevent or reduce cardiac dysfunction. For example,short-lived FAB antibody fragments are used when shorter durations oftreatment are desired. Alternatively, full-length antibodies that have alonger half-life in serum can be used, with limited dosing over 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, inclusive of all days in-between.Standard tests of cardiac function can be used to monitor progress andadjust dosing as necessary, along with assessment of relevant biomarkersdiscussed above. Limited duration of treatment adds to the safety ofthis approach.

Fibrosis of the liver is implicated in the pathology of numerous hepaticdiseases. As previously noted, fibrosis occurs as a complication ofhaemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viralhepatitis, bile duct obstruction, exposure to toxins, and metabolicdisorders. Left unchecked, hepatic fibrosis progresses to cirrhosis(defined by the presence of encapsulated nodules), liver failure, anddeath. The chronic insults to the liver from such sources as parasitesand viral infection (e.g. HBV, HCV, HIV, schistosomiasis) or the longterm stress from alcohol consumption inevitably result in remodeling ofthe liver, presumably to encapsulate the damaged area and protect theremaining liver tissue from damage. (Li and Friedman, Gastroenterol.Hepatol. 14:618-633, 1999). Liver fibrosis results in extracellularmatrix changes, including 3-10 fold increases in total collagen contentand replacement of the low density basement membrane with high-densitymatrix, which impair the metabolic and synthesis function ofhepatocytes, hepatic stellate cells and endothelial cells. (Girogescu,M., Non-invasive Biochemical Markers of Liver Fibrosis, J.Gastrointestin. Liver Dis., 15(2): 149-159 (2006)). The compounds of theinstant invention are thus useful for the prevention, treatment, and/oramelioration of fibrotic liver diseases, and such use is contemplatedherein by inhibition of LOX/LOXL2.

Like liver fibrosis, kidney fibrosis can result from various diseasesand insults to the kidneys. Examples of such diseases and insultsinclude chronic kidney disease, metabolic syndrome, diabetes andresultant glomerular nephritis. It has become recognized that metabolicsyndrome is a cluster of abnormalities including diabetic hallmarks suchas insulin resistance as well as central or visceral obesity andhypertension. In nearly all cases, dysregulation of glucose results inthe stimulation of cytokine release and upregulation of extracellularmatrix deposition. Additional factors contributing to chronic kidneydisease, diabetes, metabolic syndrome, and glomerular nephritis includehyperlipidemia, hypertension, and proteinuria, all of which result infurther damage to the kidneys and further stimulate the extracellularmatrix deposition. Thus, regardless of the primary cause, insults to thekidneys result in kidney fibrosis and the concomitant loss of kidneyfunction. (Schena, F. and Gesualdo, L., Pathogenic Mechanisms ofDiabetic Nephropathy, J. Am. Soc. Nephrol., 16: S30-33 (2005);Whaley-Connell, A., and Sower, J. R., Chronic Kidney Disease and theCardiometabolic Syndrome, J. Clin. Hypert., 8(8): 546-48 (2006)). Thecompounds of the instant invention are thus useful for the prevention,treatment, and/or amelioration of fibrotic kidney diseases (chronickidney disease, diabetic nephropathy, glomerular nephritis, metabolicsyndrome), and such use is contemplated herein.

Fibrosis of the lung includes many syndromes and diseases. Exemplarydiseases include Idiopathic pulmonary fibrosis (IPF), IdiopathicInterstitial Pneumonia, and Acute Respiratory Distress Syndrome (ARDS).The pathogenesis of most lung fibroses, including the aforementioneddiseases are not well understood, however all are characterized by aninflux of inflammatory cells and a subsequent increase in the synthesisand deposition of collagen-rich extracellular matrix. (Chua et al., AmJ. Respir. Cell. Mol. Biol., 33:9-13 (2005); Tzortzaki et al., J.Histochem. & Cytochem., 54(6): 693-700 (2006); Armstrong et al., Am. J.Respir. Crit. Care Med., 160: 1910-1915 (1999)). Given the identifiedrole of increased collagen and extracellular matrix deposition in lungfibroses, the compounds of the present invention are useful for theprevention, treatment, and/or amelioration of lung fibroses by theinhibition of LOX/LOXL2.

Scleroderma is an autoimmune disorder, in which there is anoverproduction of abnormal collagen. This excess collagen accumulatesthroughout the body, causing hardening (sclerosis), scarring (fibrosis),and other damage. The damage may affect the appearance of the skin, orit may involve only the internal organs. The symptoms and severity ofscleroderma vary from person to person. Given the identified role ofincreased collagen in scleroderma, the compounds of the presentinvention are useful for the prevention, treatment, and/or ameliorationof scleroderma by the inhibition of LOX/LOXL2.

Abnormal angiogenesis that can be treated or prevented by using thisinvention include those abnormal angiogenesis accompanying rheumatoidarthritis, ischemic-reperfusion related brain edema and injury, corticalischemia, ovarian hyperplasia and hypervascularity, (polycystic ovarysyndrome), endometriosis, psoriasis, diabetic retinopaphy, and otherocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplastic), muscular degeneration, corneal graftrejection, neuroscular glaucoma and Oster Webber syndrome.

Diseases associated with abnormal angiogenesis require or inducevascular growth. For example, corneal angiogenesis involves threephases: a pre-vascular latent period, active neovascularization, andvascular maturation and regression. The identity and mechanism ofvarious angiogenic factors, including elements of the inflammatoryresponse, such as leukocytes, platelets, cytokines, and eicosanoids, orunidentified plasma constituents have yet to be revealed.

Anti-LOX antibodies and anti-LOXL2 antibodies described herein can beused for treating diseases associated with undesired or abnormalangiogenesis. The method comprises administering to a patient sufferingfrom undesired or abnormal angiogenesis a LOX/LOXL inhibitor incombination with anti-neoplastic agent or anti-angiogenic agent that isnot said LOX/LOXL inhibitor. The particular dosage of these agentsrequired to inhibit (partially or completely) angiogenesis and/orangiogenic diseases can depend on the severity of the condition, theroute of administration, and related factors that can be decided by theattending physician. Generally, accepted and effective daily doses arethe amount sufficient to effectively inhibit angiogenesis and/orangiogenic diseases.

According to this embodiment, the pharmaceutical formulations of thepresent invention can be used to treat a variety of diseases associatedwith undesirable angiogenesis such as retinal/choroidalneuvascularization and corneal neovascularization. Examples ofretinal/choroidal neuvascularization include, but are not limited to,Bests diseases, myopia, optic pits, Stargarts diseases, Pagets disease,vein occlusion, artery occlusion, sickle cell anemia, sarcoid, syphilis,pseudoxanthoma elasticum carotid abostructive diseases, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, diabeticretinopathy, macular degeneration, Bechets diseases, infections causinga retinitis or chroiditis, presumed ocular histoplasmosis, parsplanitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications, diseases associatedwith rubesis (neovascularization of the angle) and diseases caused bythe abnormal proliferation of fibrovascular or fibrous tissue includingall forms of proliferative vitreoretinopathy. Examples of cornealneuvascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, polyarteritis,Wegener sarcoidosis, Scleritis, periphigoid radial keratotomy,neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections and Kaposi sarcoma.

In yet another embodiment, the pharmaceutical formulations of thepresent invention can be used for treating chronic inflammatory diseasesassociated with abnormal angiogenesis. The method comprisesadministering to a patient suffering from a chronic inflammatory diseaseassociated with abnormal angiogenesis a LOX/LOXL2 inhibitor incombination with anti-neoplastic agent or anti-angiogenic agent that isnot said LOX/LOXL2 inhibitor. The chronic inflammation depends oncontinuous formation of capillary sprouts to maintain an influx ofinflammatory cells. The influx and presence of the inflammatory cellsproduce granulomas and thus, maintains the chronic inflammatory state.Inhibition of angiogenesis using the compounds described herein canprevent the formation of the granulosmas, thereby alleviating thedisease. Examples of chronic inflammatory disease include, but are notlimited to, inflammatory bowel diseases such as Crohn's disease andulcerative colitis, psoriasis, sarcoidois, and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis are characterized by chronic inflammation and angiogenesis atvarious sites in the gastrointestinal tract. For example, Crohn'sdisease occurs as a chronic transmural inflammatory disease that mostcommonly affects the distal ileum and colon but may also occur in anypart of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea. Theseinflammatory bowel diseases are generally caused by chronicgranulomatous inflammation throughout the gastrointestinal tract,involving new capillary sprouts surrounded by a cylinder of inflammatorycells. Inhibition of angiogenesis by the pharmaceutical formulations ofthe present invention should inhibit the formation of the sprouts andprevent the formation of granulomas. The inflammatory bowel diseasesalso exhibit extra intestinal manifestations, such as skin lesions. Suchlesions are characterized by inflammation and angiogenesis and can occurat many sites other the gastrointestinal tract Inhibition ofangiogenesis by the pharmaceutical formulations of the present inventionshould reduce the influx of inflammatory cells and prevent the lesionformation. Therefore, provided herein is a method of treating aninflammatory bowel disease, comprising administering to a subject inneed thereof an effective amount of an anti-LOX antibody or ananti-LOXL2 antibody; and optionally, a pharmaceutically acceptablecarrier or excipient.

Sarcoidois, another chronic inflammatory disease, is characterized as amultisystem granulomatous disorder. The granulomas of this disease canform anywhere in the body and, thus, the symptoms depend on the site ofthe granulomas and whether the disease is active. The granulomas arecreated by the angiogenic capillary sprouts providing a constant supplyof inflammatory cells. By using the pharmaceutical formulations of thepresent invention to inhibit angiogenesis, such granulomas formation canbe inhibited. Psoriasis, also a chronic and recurrent inflammatorydisease, is characterized by papules and plaques of various sizes.Treatment using the pharmaceutical formulations of the present inventionshould prevent the formation of new blood vessels necessary to maintainthe characteristic lesions and provide the patient relief from thesymptoms. Therefore, provided herein is a method of prevent theformation of new blood vessels necessary to maintain the characteristiclesions and provide the patient relief from the symptoms, comprisingadministering to a subject in need thereof an effective amount of ananti-LOX antibody or an anti-LOXL2 antibody; and optionally, apharmaceutically acceptable carrier or excipient.

Rheumatoid arthritis (RA) is also a chronic inflammatory diseasecharacterized by non-specific inflammation of the peripheral joints. Itis believed that the blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis can actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Treatment using thepharmaceutical formulations of the present invention alone or inconjunction with other anti-RA agents may prevent the formation of newblood vessels necessary to maintain the chronic inflammation and providethe RA patient relief from the symptoms. Other anti-RA agents areconventional and known in the art. Therefore, provided herein is amethod of preventing or treating RA, comprising administering to asubject in need thereof an effective amount of an anti-LOX antibody oran anti-LOXL2 antibody; and optionally, one or more other anti-RAagents.

In addition to the use of the anti-LOX antibodies or anti-LOXL2antibodies alone in the treatment of the indications described above,combination therapy is also contemplated herein. The methods providedherein can further include administering an anti-cancer agent ortreatment to the patient.

Provided herein is a method of treating any of the indications describedabove by administering an anti-LOX antibody and an anti-LOXL2 antibody.

In one aspect, this invention features methods for inhibiting theinvasiveness and metastasis of tumor cells, by contacting the cells withat least one cytotoxic agent and at least one anti-LOX antibody oranti-LOX2 antibody. In general, the method includes a step of contactingmetastatic tumor cells with an amount of at least one cytotoxic agentand at least one anti-LOX antibody or anti-LOX2 antibody, which, incombination, is effective to reduce or inhibit the invasiveness ormetastatic potential of the cell. Alternatively, according to thepresent invention, an anti-LOX antibody or an anti-LOXL2 antibody can becombined with a chemotherapeutic agent to sensitize tumor cells (e.g.,transition from the EMT state to the MET state) to killed by thechemotherapeutic agent, thus not only preventing or inhibiting tumorinvasion and metastasis but also inhibiting primary tumor growth.

Any suitable anticancer agent may also be employed in the presentmethods.

As used herein the term “chemotherapeutic agent” or “chemotherapeutic”(or “chemotherapy,” in the case of treatment with a chemotherapeuticagent) is meant to encompass any non-proteinaceous (i.e., non-peptidic)chemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingsynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (articularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, foremustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicin phiI1,see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubincin(Adramycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as demopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replinisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids,e.g., paclitaxel (TAXOL™, Bristol Meyers Squibb Oncology, Princeton,N.J.) and docetaxel (TAXOTERE™., Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone;vancristine; vinorelbine (Navelbine™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in the definition of “chemotherapeutic agent” are anti-hormonalagents that act to regulate or inhibit hormone action on tumors such asanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including Nolvadex™), raloxifene,droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and toremifene (Fareston™); inhibitors of the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (Megace™), exemestane, formestane, fadrozole, vorozole(Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In one non-limiting example, this invention includes methods forsynergistically inhibiting the invasiveness and metastasis of tumorcells, by contacting the cells with at least cisplatin and at least oneanti-LOX antibody (see FIG. 18). One practicing methods described hereinwould understand that an anti-LOX2 antibody could be used in suchmethods.

In one embodiment, the anti-neoplastic agent in combination with theLOX/LOXL modulator is a tyrosine kinase inhibitor. For example, ZD1839(Iressa™ of AstraZeneca K.K.) shows a competitive effect for ATP in ATPbinding site of EGFR (epidermal growth factor receptor) tyrosine kinase,and inhibits tyrosine kinase activity by inhibiting autophosphorylationof tyrosine kinase. As a result, the anticancer effect is expressed byblocking an EGFR-equipping signal transduction (ligands such asepidermal growth factor (EGF) are bound to the extracellular domain ofEGFR, followed by activation of EGFR tyrosine kinase in theintracellular domain, causing not only autophosphorylation of EGFR butalso phosphorylation of various intracellular target proteins, thentransducing a proliferation signal from the cell surface to nucleus,then transducing the proliferation signals from the cancer cell surfaceto nucleus, and resulting in proliferation, infiltration, metastasis,angiogenesis of cancer cells) in association with proliferation,infiltration, differentiation and metastasis. IMC-C225 or cetuximab(Erbitux™) which is an EGFR-targeting monoclonal antibody) recognizesthe receptor part of EGFR on a cell membrane surface and inhibits theautophosphorylation of EGFR thereby inhibiting the tyrosine kinaseactivity. Herceptin is a monoclonal antibody against Her2/Neu which ishomologous to EGFR, and imatinib mesylate (GLEEVEC™, formerly STI-571)can inhibit both tyrosine kinase activities of BCR-Abl and c-kit(non-patent document No. 2). Sorafenib (Nexavar™) is a small molecularinhibitor of Raf kinase, PDGF (platelet-derived growth factor), VEGFreceptor 2 & 3 kinases and c-Kit.

As used herein, monoclonal antibodies against tumor antigens areantibodies elicited against antigens expressed by tumors and leukemiccells, preferably tumor-specific antigens. The monoclonal antibody alsoincludes fully human and humanized antibody.

Other examples of therapeutic antibodies for cancer therapy includeTrastuzumab (HERCEPTIN™; Overexpression of HER2 protein is associatedwith more aggressive disease and poorer prognosis in the clinic);Rituximab (RITUXAN™) that is raised against CD20 on lymphoma cells andselectively deplete normal and maligant CD20+ pre-B and mature B cells;Alemtuzumab (CAMPATH™), a monoclonal antibody that specifically targetsCD52 antigen that is found on B and T lymphocytes and used for thetreatment of chronic lymphocytic leukemia (CLL) and lymphoma; andGemtuzumab zogamicin (MYLOTARG™), an antibody conjugate that combines aspecific antibody against CD33 with a chemotherapeutic drug (zogamicin)and is indicated for the treatment of relapsed adult acute myelocyticleukemia.

In another embodiment, anti-angiogenic agent is combined with a LOX/LOXLinhibitor to treat cancer and other diseases associated with abnormal orundesirable angiogenesis. Examples of anti-angiogenic agents include,but are not limited to, retinoid acid and derivatives thereof,2-methoxyestradiol, ANGIOSTATIN™, ENDOSTATIN™, suramin, squalamine,tissue inhibitor of metalloproteinase-I, tissue inhibitor ofmetalloproteinase-2, plasminogen activator inhibitor-1, plasminogenactivator inhibitor-2, cartilage-derived inhibitor, paclitaxel, plateletfactor 4, protamine sulphate (clupeine), sulphated chitin derivatives(prepared from queen crab shells), sulphated polysaccharidepeptidoglycan complex (sp-pg), staurosporine, modulators of matrixmetabolism, including for example, proline analogs((I-azetidine-2-carboxylic acid (LACA), cishydroxyproline,d,I-3,4-dehydroproline, thiaproline, α-dipyridyl, β-aminopropionitrilefumarate, 4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone; methotrexate,mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chimp-3,chymostatin, β-cyclodextrin tetradecasulfate, eponemycin; fumagillin,gold sodium thiomalate, d-penicillamine (CDPT),beta.-1-anticollagenase-serum, alpha.2-antiplasmin, bisantrene,lobenzarit disodium, n-2-carboxyphenyl-4-chloroanthronilic acid disodiumor “CCA”, thalidomide; angiostatic steroid, cargboxynaminolmidazole;metalloproteinase inhibitors such as BB94. Other anti-angiogenesisagents include antibodies, preferably monoclonal antibodies againstthese angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms,VEGF-C, HGF/SF and Ang-1/Ang-2. Ferrara N. and Alitalo, K. “Clinicalapplication of angiogenic growth factors and their inhibitors” (1999)Nature Medicine 5:1359-1364.

Exemplary anti-fibrotic agents include, but are not limited to thecompounds such as β-aminoproprionitrile (BAPN), as well as the compoundsdisclosed in U.S. Pat. No. 4,965,288 to Palfreyman, et al., issued Oct.23, 1990, entitled “Inhibitors of lysyl oxidase, relating to inhibitorsof lysyl oxidase and their use in the treatment of diseases andconditions associated with the abnormal deposition of collagen; U.S.Pat. No. 4,997,854 to Kagan, et al., issued Mar. 5, 1991, entitled“Anti-fibrotic agents and methods for inhibiting the activity of lysyloxidase in situ using adjacently positioned diamine analogue substrate,”relating to compounds which inhibit LOX for the treatment of variouspathological fibrotic states, which are herein incorporated byreference. Further exemplary inhibitors are described in U.S. Pat. No.4,943,593 to Palfreyman, et al., issued Jul. 24, 1990, entitled“Inhibitors of lysyl oxidase,” relating to compounds such as2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine; as well as, e.g.,U.S. Pat. No. 5,021,456; U.S. Pat. No. 5,5059,714; U.S. Pat. No.5,120,764; U.S. Pat. No. 5,182,297; U.S. Pat. No. 5,252,608 (relating to2-(1-naphthyloxymethyl)-3-fluoroallylamine); and U.S. Patent ApplicationNo. 2004/0248871, which are herein incorporated by reference. Exemplaryanti-fibrotic agents also include the primary amines reacting with thecarbonyl group of the active site of the lysyl oxidases, and moreparticularly those which produce, after binding with the carbonyl, aproduct stabilized by resonance, such as the following primary amines:ethylenediamine, hydrazine, phenylhydrazine, and their derivatives,semicarbazide, and urea derivatives, aminonitriles, such asbeta-aminopropionitrile (BAPN), or 2-nitroethylamine, unsaturated orsaturated haloamines, such as 2-bromo-ethylamine, 2-chloroethylamine,2-trifluoroethylamine, 3-bromopropylamine, p-halobenzylamines,selenohomocysteine lactone. In another embodiment, the anti-fibroticagents are copper chelating agents, penetrating or not penetrating thecells. Additional exemplary compounds include indirect inhibitors suchcompounds blocking the aldehyde derivatives originating from theoxidative deamination of the lysyl and hydroxylysyl residues by thelysyl oxidases, such as the thiolamines, in particular D-penicillamine,or its analogues such as 2-amino-5-mercapto-5-methylhexanoic acid,D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulphinate,2-acetamidoethyl-2-acetamidoethanethiol sulphanate,sodium-4-mercaptobutanesulphinate trihydrate.

The present methods can be performed on cells in culture, e.g., in vitroor ex vivo, or can be performed on cells present in a subject, e.g., aspart of an in vivo therapeutic protocol. The therapeutic regimen can becarried out on a human or on other animal subjects. The anti-LOXantibody or anti-LOX2 antibody provided herein can be administered inany order relative to the chemotherapeutic agent. Sometimes, theanti-LOX antibody or anti-LOX2 antibody and the agent are administeredsimultaneously or sequentially. They can be administered at differentsites and on different dosage regimens. The enhanced therapeuticeffectiveness of the combination therapy of the present inventionrepresents a promising alternative to conventional highly toxic regimensof anticancer agents. Chemotherapeutic agents to be employed in suchmethods have been described in more detail above.

EXAMPLES

The application may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary embodiments ofthe application. The following examples are presented in order to morefully illustrate embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the application.While certain embodiments of the present application have been shown anddescribed herein, it will be obvious that such embodiments are providedby way of example only. Numerous variations, changes, and substitutionswill now occur to those skilled in the art without departing from theinvention. It should be understood that various alternatives to theembodiments described herein may be employed in practicing the methodsdescribed herein.

Example 1 Methods of Generating Murine Monoclonal Anti-LOX andAnti-LOXL2 Antibodies

Mice (BALB/c (00467)) were injected subcutaneously (s.c.), 5 times at2-3 week intervals with 0.05 mg antigen (Ag) in an adjuvant formulation.For peptide Ags, peptides were conjugated to bovine serum albumin andformulated in Freunds Adjuvant (FA) prior to immunization. For proteinAgs, the protein was formulated in Alhydrogel-Muramyl Dipeptide(ALD/MDP) adjuvant.

Mice were injected with Ag formulated in PBS, each day for 3 days via acombination of s.c., intraperitoneally (i.p.) and intravenous (i.v.)routes, 0.05 to 0.1 mg/route.

Cells from the spleen and lymph nodes of the mice were isolated andfused with P3X63-Ag8.653 myeloma cells using 50% polyethylene glycol.

Cells were cultured and a hybridoma library of HAT-selected cells wasisolated essentially as described in Kenney, et al. (“Production ofmonoclonal antibodies using a secretion capture report web.”Biotechnology 13:787-790, 1995).

The hybridoma library was cloned using a fluorescent activated cellsorter with automatic cell deposition unit.

Single viable cells were sorted into 96-well plates based upon theanalysis criteria of forward-scatter, side-scatter and propidium iodidefluorescence as described by Kenney et al.

Sera and supernatants were screened by enzyme linked immunosorbant assay(ELISA) using Ag-coated microtiter plate wells, which were thenincubated with mouse plasma or hybridoma supernatant, followed by goatmouse IgG (Fc-specific) antibody-HRP conjugate, followed by TMBsubstrate solution and stop reagent.

The plate wells were washed to remove unbound antibody or antigenbetween all incubations and results determined.

VH and VL amino acid sequences of an anti-LOXL2 murine monoclonalantibody identified using the described methods are provided in FIG. 6Aand FIG. 6B, respectively. For each variable region, signal peptides areshown in italics, CDRs are underlined and the beginning of the constantframework is shown in bold font.

A VH amino acid sequence of an anti-LOX murine monoclonal antibodyidentified using the described methods is provided in FIG. 7A. Two VLamino acid sequences of anti-LOX murine monoclonal antibodies identifiedusing the described methods are provided in FIGS. 7B and 7C. For eachvariable region, signal peptides are shown in italics, CDRs areunderlined.

Example 2 Anti-LOXL2 Antibodies were Screened Using a Protein Screen BUpdate to Assess Enzymatic Activity of LOXL2

Antibody candidates were initially chosen based on ELISA point tests.ELISA on multiple antigens was performed by Antibody Solutions andantibodies showing strong ELISA signal in the antigen of interest wereselected for further characterization in enzymatic assays. LOXL2produces hydrogen peroxide when the substrate 1,5-diaminopentane isdeaminated and the enzyme regenerated.

Antibodies were assessed for their ability to inhibit enzymatic activityusing a biochemical assay that couples the production of peroxide(liberated by LOXL2) to HRP and measuring the conversion of amplex redto a fluorescent product. Antibody hybridoma supernatant (10 μL) wasadded to 40 μL enzyme mixture (62.5 mM sodium borate pH 8.0, 5 units/mLHRP, 125 nM LOXL2, 10 ppm antifoam) and incubated at room temperaturefor 1 hour in a 96 well full area black plate. Enzymatic reaction wasstarted with the addition of 50 μL of substrate solution (50 mM sodiumborate, 100 μM amplex red reagent, 20 mM 1,5-diaminopentane (DAP), 10ppm antifoam) and read in a Molecular Devices M5 plate reader at 37° C.The plate reader was configured to read fluorescence (ex=544 nm, em=590nm) in kinetics mode for 1 hour. Data was recorded as the slope of thefluorescence response to time. These slopes were compared to a controlin which hydridoma media was added to the enzyme mixture. Slopes lessthan that of control were considered inhibitors.

Antibodies M1 (asc), M4, M11, M1, M13, M22, M16, M19, M20, M20 (asc) andM25 test antibodies were tested against BAPN (a competitive inhibitor ofLOXL2) as a positive control and LOXL2 as a negative control (see FIG.8).

One anti-LOXL2 antibody was designated AB0023. Anti-LOXL2 antibodiesrepeated inhibitory activity observed in 10 ml preparation materials inthe enzymatic assay. Inhibition was also repeated in cell-based assays(see below). Sequence analysis confirmed that the amino acid sequencesof M01, M16, M19 and M20 are identical.

Example 3 Anti-LOXL2 Antibody AB0023 and Enzymatic Activity

Enzymatic Activity of anti-LOXL2 antibodies can be assessed and IC50sdetermined.

M1, M1 (asc), M20 and M20 (asc) were assessed in the presence of 25 nMLOXL2 and 15 mM 1.5 DAP over increasing concentrations of antibody.

IC50 Determinations

Dose responses on selected antibodies were carried out against LOXL2using the coupled enzymatic assay described above. A dilution series ofantibody was created in PBST (0.01% tween-20) and 10 μL of this wasadded to 40 μL of enzyme mixture (62.5 mM sodium borate pH 8.0, 5units/mL HRP, 125 nM LOXL2, 10 ppm antifoam) and incubated at roomtemperature for 1 hour in a 96 well full area black plate. Enzymaticreaction was started with the addition of 50 μL of substrate solution(50 mM sodium borate, 100 μM amplex red reagent, 20 mM1,5-diaminopentane, 10 ppm antifoam) and read in an M5 plate readerusing conditions described above. The slopes of the fluorescenceresponse as a function of time were plotted against antibodyconcentration and the data was fit to a four parameter fit using GraFit.The midpoint of this plot is the apparent IC50 and is the concentrationat which fifty percent of the total response is inhibited.

Ab0023 was found to be a partial inhibitor of LOXL2 enzymatic activitywith an apparent IC50 of approximately 30 nM (see FIG. 9).

Based upon the use of a partial inhibitor in clinic for therapeutictreatment (i.e., Nevirapine—an approved HIV-1 drug described by Spenceet al. (1995) Science 267), a partial inhibitor of LOXL2 can also beused in therapeutic applications.

Example 4 Anti-LOXL2 Antibody AB0023 is a Non-Competitive Inhibitor

The activity of anti-LOXL2 antibody AB0023 was assessed over increasingconcentrations of 1,5 DAP and over increasing concentrations of antibody(1 μM, 0.005 μM, 0.050 μM, and 0.300 μM).

Mode of Inhibition

Mode of inhibition of antibodies against LOXL2 was conducted using themodel described below. In these experiments, the dependence of thesteady state rate on the concentration of 1,5-diaminopentane wasmonitored under increasing concentrations of antibody. The purpose wasto assess whether the K_(m) for substrate, k_(cat) or both change in thepresence of antibody. Collected data was analyzed globally with Grafitusing the model shown in figure below. E represents enzyme, S representssubstrate, A represents antibody, and P represents product. Parameter αdescribes the effect of the compound on substrate affinity. An α valueequal to one describes a situation in which the compound binds equallywell the free enzyme and the enzyme-substrate complex (non-competitiveinhibition like). Values less than one describe an interaction in whichthe compound binds the enzyme-substrate complex (uncompetitiveinhibition like). Values greater than one correspond to the compoundbinding the free enzyme better than the enzyme-substrate complex(competitive inhibition like). The β value describes the effect of themodulator on the rate of the enzyme. Inhibitors have values less thanone (for a complete inhibitor (β=0) and activators have values greaterthan one. K_(A) is the dissociation constant of the compound, K_(s) isthe Michaelis constant for the substrate and k is the catalytic rate ofthe enzyme. The steady state rates were determined from the slope of thefluorescence response as a function of time as described above (IC50determination). Data was plotted as the dependence of steady state rateon the concentration of substrate (1,5-diaminopentane) at several fixedconcentrations of antibody and analyzed with GraFit.

Anti-LOXL2 antibody AB0023 was determined to be a non-competitiveinhibitor based on the following results: α=1, K_(i)=0.067 and β=0.5(see FIG. 10).

Example 5 Kinetic Measurement of AB0023 Antibody Binding to LOXL2 bySurface Plasmon Resonance

Binding affinity and off-rate of AB0023 were assessed via SurfacePlasmon Resonance (SPR).

Binding affinities were measured using a Bio-Rad ProteOn instrumentthermostated to 25° C. The binding affinities were determined using twomethods, using amine coupling; one in which the antibody was immobilizedand the antigen (LOXL2) was added, and another in which the antigen(LOXL2) was immobilized and antibody was added. Antibody or antigen wasimmobilized on a GLC chip using at 1:1 ratio of NHS to EDC provided withthe ProteOn immobilization kit. Chip was first activated with NHS/EDC amixture and then antigen or antibody at 1 μg/mL in acetate buffer pH 4.5was flowed over activated surface to couple. This typically yielded acoupling of about 500 RU's. The activated chip surface was then cappedwith the addition of 1M ethanolamine. Coupled chips were stored at 4° C.and regenerated with 50 mM sodium hydroxide.

Dissociation constants were determined by probing the coupled chip witha dilution series of antibody or antigen in PBST (0.05% Tween-20). Datawas acquired on all six channels available on the ProteOn using anon-coupled channel as a reference. Collected data was analyzed usingProteOn manager software from Bio-Rad.

AB0023 was found to bind tightly to LOXL2 and release slowly. Kd wasestimated to be 0.1-1.0 nM. Furthermore, AB0023 was found to have thefollowing characteristics: k_(on)=1.68×10⁶ M⁻¹s⁻¹, k_(off)=1.17×10⁻⁴s⁻¹,K_(D)=0.69 nM and t_(1/2)=98.7 minutes. See FIG. 11.

Example 6 Domain Mapping was Conducted and AB0023 was Found to Bind tothe SRCR3-4 Domain Materials and Methods

All plates were obtained from Corning. Secondary antibody and Picosubstrate were obtained from Pierce. Horse radish peroxidase (HRP) wasobtained from Sigma. All ProteOn reagents were obtained from Bio-Rad.LOXL2 was obtained from R&D systems. Antibodies used in this study wereproduced at Antibody Solution or via ascites from Aragen Biosciences.All other reagents were of the highest quality possible.

Binding Via ELISA

Binding of antibody to LOXL2 was determined using a luminescence basedELISA. White Corning plates were coated with 0.1 μg/mL of LOXL2 orantigen of interest in 50 mM borate buffer (pH 8.0) overnight at 4° C.Plates were washed using BioTek plate washer and blocked with 5% skimmilk in PBST (0.05% tween-20) for 1 hour at room temperature. Plateswere washed with PBST (0.05% tween-20) and then used immediately orstored at 4° C. in dessicator for future use. The antibody body to betested was serially diluted in PBST (0.01% tween-20) and 100 μL of eachdilution was added per well. Plates were incubated with test article for1 hour at room temperature and then washed with PBST (0.05% Tween-20).Detection antibody (anti-mouse HRP conjugate) was diluted 16,000 fold in5% skim milk in PBST (0.05% Tween-20) and 100 μL was applied per well.Plates were incubated for 1 hour with detection antibody and then washedwith PBST (0.05% PBST). Signal was detected using the SuperSignal ELISApico chemiluminescent substrate from Pierce following the manufacturer'sinstructions. Luminescence was measure using a Molecular Devices M5plate reader with an integration time of 500 ms capturing allwavelengths. Data was background corrected and the dependence ofluminescence signal to antibody concentration was fit using the Langmuirisotherm equation using the GraFit program. In instances where theantigen concentration was similar to the dissociation constant thequadratic equation of tight binding was used. Reported dissociationvalues were obtained from the fits to these equations; were PLrepresents the signal of the bound complex, B_(max) is that maximalbinding, K_(D) is the dissociation constant and L is the ligandconcentration.

Langmuir Isotherm equation:

$\lbrack{PL}\rbrack = {\frac{B_{\max}*\lbrack L\rbrack}{K_{D}*\lbrack L\rbrack}.}$

Tight binding equation:

$\lbrack{PL}\rbrack = {B_{\max}*{\frac{( {\lbrack P\rbrack_{T} + \lbrack L\rbrack_{T} + K_{D}} ) - \sqrt{( {\lbrack P\rbrack_{T} + \lbrack L\rbrack_{T} + K_{D}} )^{2} - {{4\lbrack P\rbrack}_{T}\lbrack L \}}_{T}}}{{2\lbrack P\rbrack}_{T}}.}}$

AB0023 was tested against MCD-LOXL2, LOXL2 (R&D), SRCR1-2 and SRCR 1-4.AB0023 was found to bind to the SRCR3-4 domain (see FIG. 12).

Example 7 Inhibition of Migration/Invasion in Collagen I and Collagen IVand Inhibition of Cell Growth

Cell based assays were conducted to assess binding of AB0023 to bindsubstrate (i.e., collagen).

Briefly, AB0023 was scaled up from various samples prior to testing.

Cultrex 96-well collagen I and collagen IV cell invasion kits (Trevigen,Gaithersburg, Md.) were used for anti-sera/antibody supernatantscreenings. MDA MB 231 cells were serum-deprived 24 hours prior to assayset up. On day of set up, collagen I and collagen IV coated plates weremade at least 4 hours prior to invasion assay set up (not longer than 8hours prior). Collagen I and collagen IV plates were coated according tomanufacturer's instructions. Cells were plated at 20,000 cells per wellin 95 μls serum-free media in the upper chamber of the plate. Onehundred fifty (150) μls of media containing 10% FBS and 1× L-glutaminewas aliquoted into the lower chambers of the plate. Using amulti-channel pipette, 5 μls of each anti-sera was placed in the upperchambers of the plate. The anti-sera and cell mixture was carefullymixed up and down once with the pipette. Plates were incubated at 37° C.with 5% CO₂ for 48 hours.

After 48 hours, the plates were ready to be read. The cell dissociationsolution containing calcein AM was made according to manufacturer'sinstructions. The plates were also washed and disassembled according tomanufacturer's instructions. 125 μls of cell dissociation solutioncontaining calcein AM was added to the lower chambered wells and theplates were placed at 37° C. for 30 minutes. After 30 minutes the sidesof the plates were tapped to loosen the cells, and the plates wereplaced in incubator for another 30 minutes at 37° C. The plates werethen disassembled and the lower plate was placed into the plate reader(SpectraMax M5, Molecular Devices, Sunnyvale, Ca) with settings:Flourescence, 485-520 emission, top read, black opaque plate,sensitivity at 30.

Consistent inhibition of migration/invasion was observed in collagen I(FIG. 13) and collagen IV, from supernatents through 10 mL preparationmaterial and scaled up 100 mL preparations and ascites.

Cell Adhesion Assays

MDA-MB231 cells were plated in 15 cm² plates and grown in 4.5 g/Lglucose containing DMEM (10% FBS and 2 mM L-Glutamine) so that they wereconfluent on the day of the assay. The media was aspirated and the cellswere washed 2 times with 10 ml 1 mM EDTA PBS per plate. Cells wereremoved from plates by incubating with another 10 ml 1 mM EDTA PBS for 5minutes at 37° C. in a biosafety cabinet and subsequently pipetting thecells off of the plate in the EDTA PBS solution. Cell concentration wasdetermined and enough cells for assay (50 k/well plus extra forpipetting) were spun-down in 15 ml conical tube. Cell pellet wasdispersed in pre-warmed serum-free DMEM to 500K cells/ml and CuCl₂ wasadded to 1 μM final concentration. One hundred (100) μl/well of cellsuspension was pipetted into a U-bottom style 96 well tissue cultureplate containing 10 μl of appropriate mAb dilution. The cellsuspension/mAb mixture was left to incubate for 10 minutes at roomtemperature in the dark. One hundred (100) μl/well of re-suspendedcells/mAb mixture was then transferred to collagen IV coated 96 wellplates (BD Biocoat, BD Biosciences). Plates were incubated at 37° C. ina biosafety cabinet for 1 hour. Wells were then aspirated and washedgently two times with 200 μl DPBS (Mediatech) to remove cell that hadnot adhered. One hundred (100) μl of 10 μM final concentrationCalcein-AM (BD Biosciences) in DPBS was then added to each well to stainthe cells that remained. Plates were incubated at 37° C. in a biosafetycabinet for 1 hour. Plates were read on a Molecular Devices M5 platereader at 494/517 (excitation/emission). Percent (%) adhesion wascalculated by normalizing to PBS or un-related antibody controls.

Using this assay, partial inhibition of adhesion was observed uponexposure of cells to AB0023 antibody.

Cell Growth

Anti-LOXL2 antibodies inhibited cell growth of four cell lines: 231 is abreast cancer cell line, BT549 is a breast cancer cell line, HT1080 is afibrosacrcoma, and BxPC3 is a prostate cancer cell line (FIG. 17). Thus,the antibody is effective in inhibiting growth of cancers of differentorigins.

Example 8 AB0023 Inhibition of EMT-Like Change

Epithelial to Mesenchymal changes were assessed usingimmunohistochemistry.

To detect whether a cell is in an EMT or mesenchymal-to-epithelialtransition (MET) state, cells were stained with antibodies specific tocellular protein markers for epithelial or mesenchymal states such asE-cadherin, vimentin, fibronectin, and phalloidin to detect F-actin.

Rhodamine Phalloidin Staining Protocol

Cells were seeded 24 hours prior to day of staining; cells wereapproximately 80% confluent 24 hours later in an 8-chambered slide. Thenext day, the media was aspirated and the chambers were rinsed with1×PBS. Cells were then fixed with 4% Parafomaldehyde (PFA) for 20minutes at room temperature and then rinsed once with 1× PhosphateBuffered Saline (PBS). For permeabilization, the cells were treated with0.5% Saponin (JT Baker, Phillipsburg, N.J.) in PBS for 5 minutes at roomtemperature. The chambers were carefully rinsed once with 1×PBS, and a1:100 dilution of rhodamine phalloidin (Invitrogen, Carlsbad, Ca) in PBSwas added to the cells and incubated for 15 minutes at room temperature.The chambers were rinsed two times with 1×PBS and the slides weremounted with Vectashield (Vector Laboratories, Burlingame, Calif.).

E-Cadherin Staining Protocol

Cells were seeded 24 hours prior to day of staining; cells wereapproximately 80% confluent the next day in an 8-chambered slide. Thenext day, the media was aspirated and the chambers were rinsed with1×PBS. Cells were then fixed with ice cold methanol and then incubatedfor 2 minutes in −20° C. The cells were rinsed once with 1×PBX and 1μg/ml of E-cadherin Ab (Calbiochem, Gibbstown, N.J.) was added to theslide chambers. The slides were then incubated at 37° C. for 1 hour.After carefully rinsing the chambers one time with 1×PBS, the secondaryAb (anti-mouse IgG cy3 conjugated, Jackson Immuno Research, West Grove,Pa.) was added and incubated at room temperature for 30-45 minutes. Thechambers were rinsed two times with 1×PBS and mounted with Vectashield(Vector Laboratories, Burlingame, Calif.).

Conditioned media from HS-578t cells (LOXL2 high) applied to MCF-7 cells(LOXL2 low/negative). Cells were stained with rhodamine-phalloidin(F-actin, red) and Dapi (nuclei, blue).

AB0023 was found to inhibit EMT-like changes induced by conditionedmedia from tumor cells that express LOXL2 (data not shown).

Example 9 Anti-LOXL2 Antibody AB0023 Binds Matrix-Associated LOXL2Internalization and Ab Uptake Studies in Hs578t Cells.

Hs578t cells were cultured in DMEM containing 10% FBS and 1× glutamine.The cells were seeded in an 8 chamber glass slide (BD Falcon, FranklinLakes, N.J.) and allowed to adhere overnight. For low confluency, cellswere seeded at 30-40,000 cells per slide. Low confluency was used fordetection of Lox in the cytosol 24 hours later. For high confluency,cells were seeded at 100,000 cells per slide. High confluency was usedfor detection of Lox associated with the matrix and collagenapproximately 48-72 hours later.

The following day, 1 μg/ml (final concentration in regular growthmedium) of anti-Lox M64 or anti-Lox12 M20 monoclonal Ab (mAb) was addedto the chambers. For continuous uptake, the mAbs were incubated withcells at different time points: for example, 3 hours, 8 hours, or 24hours (overnight). After appropriate amount of continuous uptake, themedia was removed and the chambers were rinsed with 1×PBS. The cellswere fixed in 4% PFA (paraformaldehyde) at room temperature for 20minutes. After fixation, the cells were washed with 1×PBS at roomtemperature for 5 minutes and then quenched in 50 mM ammonium chlorideat room temperature for 10 minutes. The cells were washed again with1×PBS at room temperature for 5 minutes.

The cells were permeabilized by adding saponin buffer (0.5% Saponin/1%BSA in PBS) at room temperature for 20 minutes. The secondary detectionAb (Alexa Fluor 488 donkey anti-mouse IgG, Invitrogen, Carlsbad, Calif.)was added at room temperature in saponin buffer and the cells wereincubated for 30-45 minutes. The cells were then washed 3× in saponinbuffer. The slides were mounted with vectashield (Vector Laboratories,Burlingame, Calif.).

For detection of collagen detection, cells were incubated withanti-collagen antibody (1:50, Calbiochem anti-collagen type I Rabbitpolyclonal, Gibbstown, N.J.), one hour prior to fixing the cells with 4%PFA. Secondary Ab for collagen used is donkey anti-rabbit Cy3(ImmunoJackson Labs, West Grove, Pa.).

Analysis by immunoblotting and immunofluorescence (data not shown)indicated that LOXL2 was predominantly intracellular at low density butwas secreted at high cell density (confluent cells). LOXL2 was detectedin the media of confluent cells and also on the extracellular matrixImmunofluorescence on live cells indicated that AB0023 bound LOXL2associated with the collagen matrix.

Example 10 Anti-LOX Antibody M64 Binds LOX

The activity of anti-LOX antibody M64 was assessed over increasingconcentrations of 1,5 DAP and over increasing concentrations of antibody(see FIG. 15).

Materials and Methods

All plates were obtained from Corning. Secondary antibody and Picosubstrate were from Pierce. Amplex red reagent was from Invitrogen.Horse radish peroxidase (HRP), 1,5-diaminopentane, antifoam were fromSigma. All ProteOn reagents were from Bio-Rad. LOX was produced in houseat Arresto Biosciences. Antibodies used in this study were produced atAntibody Solution or via ascites (asc) from Aragen Biosciences. Allother reagents were of the highest quality possible.

Binding Via ELISA

Binding of antibody to LOX was determined using a luminescence basedELISA. White Corning plates were coated with 0.1 ug/mL of LOX or antigenof interest in 50 mM borate buffer (pH 8.0) overnight at 4° C. Plateswere washed using a BioTek plate washer and blocked with 5% skim milk inPBST (0.05% tween-20) for 1 hour at room temperature. Plates were washedwith PBST (0.05% tween-20) and then used immediately or stored at 4° C.in a dessicator for future use. The antibody to be tested was seriallydiluted in PBST (0.01% tween-20) and 100 uL of each dilution was addedper well. Plates were incubated with test material for 1 hour at roomtemperature and then washed with PBST (0.05% tween-20). Detectionantibody (anti-mouse HRP conjugate) was diluted 16000 fold in 5% skimmilk in PBST (0.05% tween-20) and 100 uL was applied per well. Plateswere incubated for 1 hour with detection antibody and then washed withPBST (0.05% PBST). Signal was detected using the SuperSignal ELISA picochemiluminescent substrate from Pierce following the manufacturer'sinstructions. Luminescence was measured using a Molecular Devices M5plate reader with an integration time of 500 ms capturing allwavelengths. Data was background corrected and the dependence ofluminescence signal to antibody concentration was fit using the Langmuirisotherm equation using the GraFit program. In instances where theantigen concentration was similar to the dissociation constant thequadratic equation of tight binding was used. Reported dissociationvalues were obtained from the fits to these equations; were PLrepresents the signal of the bound complex, B_(max) is that maximalbinding, K_(D) is the dissociation constant and L is the ligandconcentration.

Langmuir Isotherm Equation

Langmuir Isotherm equation:

$\lbrack{PL}\rbrack = {\frac{B_{\max}*\lbrack L\rbrack}{K_{D}*\lbrack L\rbrack}.}$

Tight binding equation:

$\lbrack{PL}\rbrack = {B_{\max}*{\frac{( {\lbrack P\rbrack_{T} + \lbrack L\rbrack_{T} + K_{D}} ) - \sqrt{( {\lbrack P\rbrack_{T} + \lbrack L\rbrack_{T} + K_{D}} )^{2} - {{4\lbrack P\rbrack}_{T}\lbrack L \}}_{T}}}{{2\lbrack P\rbrack}_{T}}.}}$

Anti-LOX antibody M64 was tested in three batches and was found to havea KD of 6.6 nM, 5.0 nM and 5.7 nM for Batch 3, Batch 4 and Batch 5,respectively (FIG. 15).

Example 11 Kinetic Measurement of M64 Antibody Binding to LOX by SurfacePlasmon Resonance

Binding affinity of M64 was assessed via Surface Plasmon Resonance(SPR).

Binding affinities were measured using a Bio-Rad ProteOn instrumentthermostated to 25° C. The binding affinities were determined using twomethods, using amine coupling; one in which the antibody was immobilizedand the antigen (LOX) was added, and another in which the antigen (LOX)was immobilized and antibody was added. Antibody or antigen wasimmobilized on a GLC chip using at 1:1 ratio of NHS to EDC provided withthe ProteOn immobilization kit. Chip was first activated with NHS/EDC amixture and then antigen or antibody at 1 μg/mL in acetate buffer pH 4.5was flowed over activated surface to couple. This typically yielded acoupling of about 500 RU's. The activated chip surface was then cappedwith the addition of 1M ethanolamine. Coupled chips were stored at 4° C.and regenerated with 50 mM sodium hydroxide.

Dissociation constants were determined by probing the coupled chip witha dilution series of antibody or antigen in PBST (0.05% tween-20). Datawas acquired on all six channels available on the ProteOn using anon-coupled channel as a reference. Collected data was analyzed usingProteOn manager software from Bio-Rad.

M64 was found to have a K_(D) of 7 nM (FIG. 16).

Example 12

Below is a listing of sequences described throughout the specification.

SEQ ID NO Sequence  1.MEWSRVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKASGYAFTYYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARNWMNFDYWGQGTTLTVSS  2.MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVSVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQFLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK  3.MGWSWVFLFLLSVTAGVHSQVQLQQSGAELVKPGASVKLSCKASGYTFRSYDINWVRQRPEQGLEWIGWIFPGDGSTKYNEKFKGKAILTTDKSSSTAYMQLSRLTSEDSAVYFCARVYYAMDYWGQGTSVTVSS  4.MKLPVRLLVMFWIPASSSDVLLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFGGSGSGTDFTLKINRVEAEDLGIYYCFQSSHIPLTFGAGTKLELKRAD  5.MKLPVRLLVMFWIPASSSDVLLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSIRFSGVPDRFGGSGSGTDFTLKINRVEAEDLGIYYCFQSSHIPLTFGAGTKLELKRAD  6.VRLRGGAYIGEGRVEVLKNGEWGTVCDDKWDLVSASVVCRELGFGSAKEAVTGSRLGQGIGPIHLNEIQCTGNEKSIIDCKFNAESQGCNHEEDAGVRCNTPAMGLQKKLRLNGGRNPYEGRVEVLVERNGSLVWGMVCGQNWGIVEAMVVCRQLGLGFASNAFQETWYWHGDVNSNKVVMSGVKCSGTELSLAHCRHDGEDVACPQGGVQYGAGVACS  7. MRFAWTVLLLGPLQLCA LVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMV G DDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY  8.ALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMV GDD PYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAY ASGCTISPY  9.DDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 10.gggcgtgatttgagccccgtttttattttctgtgagccacgtcctcctcgagggggtcaatctggccaaaaggagtgatgcgcttcgcctggaccgtgctcctgctcgggcctttgcagctctgcgcgctagtgcactgcgcccctcccgccgccggccaacagcagcccccgcgcgagccgccggcggctccgggcgcctggcgccagcagatccaatgggagaacaacgggcaggtgttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggcgcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaacgtggcgcctcgcgcgcggagaaccagacagcgccgggagaagttcctgcgctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtctcccagacctggtggccgacccctactacatccaggcgtccacgtacgtgcagaagatgtccatgtacaacctgagatgcgcggcggaggaaaactgtctggccagtacagcatacagggcagatgtcagagattatgatcacagggtgctgctcagatttccccaaagagtgaaaaaccaagggacatcagatttcttacccagccgaccaagatattcctgggaatggcacagttgtcatcaacattaccacagtatggatgagtttagccactatgacctgcttgatgccaacacccagaggagagtggctgaaggccacaaagcaagtttctgtcttgaagacacatcctgtgactatggctaccacaggcgatttgcatgtactgcacacacacagggattgagtcctggctgttatgatacctatggtgcagacatagactgccagtggattgatattacagatgtaaaacctggaaactatatcctaaaggtcagtgtaaaccccagctacctggttcctgaatctgactataccaacaatgttgtgcgctgtgacattcgctacacaggacatcatgcgtatgcctcaggctgcacaatttcaccgtattagaaggcaaagcaaaactcccaatggataaatcagtgcctggtgttctgaagtgggaaaaaatagactaacttcagtaggatttatgtattttgaaaaagagaacagaaaacaacaaaagaatttttgtttggactgttttcaataacaaagcacataactggattttgaacgcttaagtcaatcattacttggaaatttntaatgtttattatttacatcaactttgtgaattaacacagtgtttcaattctgtaatttcatatttgactcttt 11.MRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRARERGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 12.atgcgcttcgcctggaccgtgctcctgctcgggcctttgcagctctgcgcgctagtgcactgcgcccctcccgccgccggccaacagcagcccccgcgcgagccgccggcggctccgggcgcctggcgccagcagatccaatgggagaacaacgggcaggtgttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggcgcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaacgtggcgcctcgcgcgcggagaaccagacagcgccgggagaagttcctgcgctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtctcccagacctggtggccgacccctactacatccaggcgtccacgtacgtgcagaagatgtccatgtacaacctgagatgcgcggcggaggaaaactgtctggccagtacagcatacagggcagatgtcagagattatgatcacagggtgctgctcagatttccccaaagagtgaaaaaccaagggacatcagatttcttacccagccgaccaagatattcctgggaatggcacagttgtcatcaacattaccacagtatggatgagtttagccactatgacctgcttgatgccaacacccagaggagagtggctgaaggccacaaagcaagtttctgtcttgaagacacatcctgtgactatggctaccacaggcgatttgcatgtactgcacacacacagggattgagtcctggctgttatgatacctatggtgcagacatagactgccagtggattgatattacagatgtaaaacctggaaactatatcctaaaggtcagtgtaaaccccagctacctggttcctgaatctgactataccaacaatgttgtgcgctgtgacattcgctacacaggacatcatgcgtatgcctcaggctgcacaatttcaccgtattag 13.MRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRARERGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 14.gggccaggactgagaaaggggaaagggaagggtgccacgtccgagcagccgccttgactggggaagggtctgaatcccacccttggcattgcttggtggagactgagatacccgtgctccgctcgcctccttggttgaagatttctccttccctcacgtgatttgagccccgtttttattttctgtgagccacgtcctcctcgagcggggtcaatctggcaaaaggagtgatgcgcttcgcctggaccgtgctcctgctcgggcctttgcagctctgcgcgctagtgcactgcgcccctcccgccgccggccaacagcagcccccgcgcgagccgccggcggctccgggcgcctggcgccagcagatccaatgggagaacaacgggcaggtgttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggggcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaagctgggccctcgcgcgcggagaaccagacagcgccgggagaagttcctgctctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtctcccagacctggtggccgacccctactacatccaggcgtccacgtacgtgcagaagatgtccatgtacaacctgagatgcgcggcggaggaaaactgtctggccagtacagcatacagggcagatgtcagagattatgatcacagggtgctgctcagatttccccaaagagtgaaaaaccaagggacatcagatttcttacccagccgaccaagatattcctgggaatggcacagttgtcatcaacattaccacagtatggatgagtttagccacttgtacctgcttgatgccaacacccagaggagatgggctgaaggccacaaagcaagtttctgtcttgaagacacatcctgtgactatggctaccacaggcgatttgcatgtactgcacacacacagggattgagtcctggctgttatgatacctatggtgcagacatagactgccagtggattgatattacagatgtaaaacctggaaactatatcctaaaggtcagtgtaaaccccagctacctggttcctgaatctgactataccaacaatgttgtgcgctgtgacattcgctacacaggacatcatgcgtatgcctcaggctgcacaatttcaccgtattagaaggcaaagcaaaactcccaatggataaatcagtgcctggtgttctgaagtgggaaaaaatagactaacttcagtaggatttatgtattttgaaaaagagaacagaaaacaacaaaagaatttttgtttggactgttttcaataacaaagcacataactggattttgaacgcttaagtcatcattacttgggaaatttttaatgtttattatttacatcactttgtgaattaacacagtgtttcaattctgtaattacatatttgactctttcaaaaaaaaaaaaaaaaaaaaaaa 15.MRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAGRTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGPSRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHLYLLDANTQRRWAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 16.ccgcgccgctccccgttgccttccaggactgagaaaggggaaagggaagggtgccacgtccgagcagccgccttgactggggaagggtctgaatcccacccttggcattgcctggtggagactgagatacccgtgctccgctcgcctccttggttgaagatttctccttccctcacgtgatttgagccccgtttttattttctgtgagccacgtcctcctcgagcggggtcaatctggcaaaaggagtgatgcgcttcgcctggaccgtgctcctgctcgggcctttgcagctctgcgcgctagtgcactgcgcccctcccgccgccggccaacagcagcccccgcgcgagccgccggcggctccgggcgcctggcgccagcagatccaatgggagaacaacgggcaggtgttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggcgcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaagctggcgcctcgcgcgcggagaaccagacagcgccgggagaagttcctgcgctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtctcccagacctggtggccgacccctactacatccaggcgtccacgtacgtgcagaagatgtccatgtacaacctgagatgcgcggcggaggaaaactgtctggccagtacagcatacagggcagatgtcagagattatgatcacagggtgctgctcagatttccccaaagagtgaaaaaccaagggacatcagatttcttacccagccgaccaagatattcctgggaatggcacagttgtcatcaacattaccacagtatggatgagtttagccactatgacctgcttgatgccaacacccagaggagagtggctgaaggccacaaagcaagtttctgtcttgaagacacatcctgtgactatggctaccacaggcgatttgcatgtactgcacacacacagggattgagtcctggctgttatgatacctatggtgcagacatagactgccagtggattgatattacagatgtaaaacctggaaactatatcctaaaggtcagtgtaaaccccagctacctggttcctgaatctgactataccaacaatgttgtgcgctgtgacattcgctacacaggacatcatgcgtatgcctcaggctgcacaatttcaccgtattagaaggcaaagcaaaactcccaatggataaatcagtgcctggtgttctgaagtgggaaaaaatagactaacttcagtaggatttatgtattttgaaaaagagaacagaaaacaacaaaagaatttttgtttggactgttttcaataacaaagcacataactggattttgaacgcttaagtcatcattacttgggaaatttttaatgtttattatttacatcactttgtgaattaacacagtgtttcaattctgtaattacatatttgactctttcaaagaaatccaaatttctcatgttccttttgaaattgtagtgcaaaatggtcagtattatctaaatgaatgagccaaaatgactttgaactgaaacttttctaaagtgctggaactttagtgaaacataataataatgggtttatacgacagcaacgga 17.MRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 18.ggtcaatctggcaaaaggagtgatgcgcttcgcctggaccgtgctcctgctcgggcctttgcagctctgcgcgctagtgcactgcgcccctcccgccgccggccaacagcagcccccgcgcgagccgccggcggctccgggcgcctggcgccagcagatccaatgggagaacaacgggcaggtgttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggcgcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaagctggcgcctcgcgcgcggagaaccagacagcgccgggagaagttcctgcgctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtctcccagacctggtggccgacccctactacatccaggcgtccacgtacgtgcagaagatgtccatgtacaacctgagatgcgcggcggaggaaaactgtctggccagtacagcatacagggcagatgtcagagattatgatcacagggtgctgctcagatttccccaaagagtgaaaaaccaagggacatcagatttcttacccagccgaccaagatattcctgggaatggcacagttgtcatcaacattaccacagtatggatgagtttagccactatgacctgcttgatgccaacacccagaggagagtggctgaaggccacaaagcaagtttctgtcttgaagacacatcctgtgactatggctaccacaggcgatttgcatgtactgcacacacacagggattgagtcctggctgttatgatacctatggtgcagacatagactgccagtggattgatattacagatgtaaaacctggaaactatatcctaaaggtcagtgtaaaccccagctacctggttcctgaatctgactataccaacaatgttgtgcgctgtgacattcgctacacaggacatcatgcgtatgcctcaggctgcacaatttcaccgtattagaaggcaaagcaaaactcccaatggataaatcagtgcctggtgttct 19.MRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 20.ggtcaatctggcaaaaggagtgatgcgcttcgcctggaccgtgctcctgctcgggcctttgcagctctgcgcgctagtgcactgcgcccctcccgccgccggccaacagcagcccccgcgcgagccgccggcggctccgggcgcctggcgccagcagatccaatgggagaacaacgggcaggtgttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggcgcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaagctggcgcctcgcgcgcggagaaccagacagcgccgggagaagttcctgcgctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtctcccagacctggtggccgacccctactacatccaggcgtccacgtacgtgcagaagatgtccatgtacaacctgagatgcgcggcggaggaaaactgtctggccagtacagcatacagggcagatgtcagagattatgatcacagggtgctgctcagatttccccaaagagtgaaaaaccaagggacatcagatttcttacccagccgaccaagatattcctgggaatggcacagttgtcatcaacattaccacagtatggatgagtttagccactatgacctgcttgatgccaacacccagaggagagtggctgaaggccacaaagcaagtttctgtcttgaagacacatcctgtgactatggctaccacaggcgatttgcatgtactgcacacacacagggattgagtcctggctgttatgatacctatggtgcagacatagactgccagtggattgatattacagatgtaaaacctggaaactatatcctaaaggtcagtgtaaaccccagctacctggttcctgaatctgactataccaacaatgttgtgcgctgtgacattcgctacacaggacatcatgcgtatgcctcaggctgcacaatttcaccgtattagaaggcaaagcaaaactcccaatggataaatcagtgcctggtgttctgaa 21.MRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 22.Gttcagcttgctgagcctgggctcacagtaccagcctcagcgccgccgggacccgggcgccgccgtccctggtgcagccaacgcctccgcccagcagccccgcactccgatcctgctgatccgcgacaaccgcaccgccgcggcgcgaacgcggacggccggctcatctggagtcaccgctggccgccccaggcccaccgcccgtcactggttccaagctggctactcgacatctagagcccgcgaagctggcgcctcgcgcgcggagaaccagacagcgccgggagaagttcctgcgctcagtaacctgcggccgcccagccgcgtggacggcatggtgggcgacgacccttacaacccctacaagtactctgacgacaacccttattacaactactacgatacttatgaaaggcccagacctgggggcaggtaccggcccggatacggcactggctacttccagtacggtaagtacccccaagtccgctggaagcacccgtgcacctggtccccagctatgtggcttcttctcgacgtggctgcctgggcgcggcgggccccggtcctcgcagatccgacccctccccacgcgcctgcagtggcagccctggaatccagtgcaaaccgcgcgtctggcccctcctgcttccttttcacattgctttgcagtcccgggggtccccagttctcttgctgtcctccgctccactctgcagtcccggtgggcgaagggtgaggagtaagggacctagaggggtagggagttggagcggggggcgccgggttgtttcactgctgcgcccgtcgcctgctgacgtttaggtctcccagacctgg tg 23.FSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLV 24.MEWSRVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKAS GYAFTYYLIE WV KQRPGQGLEWIGVINPGSGGTNYNEKFKG KATLTADKSSSTAYMQLSSLTSDDSAV YFCAR NW MN FDYWGQGTTLTVSS 25. QVQL V QSGAEL KK PG A SVKVSCKASGYAFTYYLIEWVKQ APGQGLEWIG VINPGSG GTNYNEKFKG R ATLTADKS T STAYM E LSSL R S E DSAVYFCARNWMNFDY WGQGTT V TVSS 26. QVQL V QSGAE VKK PGASVKVSCKASGYAFTYYLIEWV R QA PGQGLEWIGVINPGSG GTNYNEKFKGR ATLTADKS T STAYM E LSSL R S E D TAVYFCARNWMNFDYWGQGTT V TVSS 27. QVQL V QSGAE VKK PG A SVKVSCKASGYAFTYYLIE WV R Q A PGQGLEWIG VINPGSG GTNYNEKFKG R AT I TADKS T STAYM ELSSL R S E D T AVYFCAR NWMNFDY WGQGTT V TVSS 28. QVQL V QSGAE VKK PG ASVKVSCKAS GYAFTYYLIE WV R Q A PGQGLEWIG VINPGSG GTNYNEKFKG RV T I TADKST STAYM E LSSL R S E D T AVY Y CAR NWMNFDY WGQGTT V TVSS 29.MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVSVTPGESVSISC RSSKSLLHSNGNT YLYWFLQRPGQSPQFLIY RMSNL AS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYY C MQHLEYPYTFGGGTKLEIK 30. DIVMTQ TPL S L SVTPG QPA SISC RSSKSLLHSNGNTYLY WFLQ KPGQSPQFLIY RM SNLAS GVPDRFSGSGSGTAFTL K ISRVEAEDVGVYYC MQHLEYPYT FGGGTKV EIK 31. DIVMTQ TPL S L SVTPG QPA SISC RSSKSLLHSNGNTYLY WFLQ KPGQSPQFLIY RM SNLAS GVPDRFSGSGSGT D FTL K ISRVEAEDVGVYYC MQHLEYPYTFGGGTK V EIK 32. DIVMTQ TPL S L SVTPG QPA SISC RSSKSLLHSNGNTYLY W Y LQ KPGQSPQFLIY RM SNLAS GVPDRFSGSGSGT D FTL K ISRVEAEDVGVYYC MQHLEYPYTFGGGTK V EIK 33. MEWSRVFIFLLSVTAGVHSQVQLQQSGAELVRPGTSVKVSCKAS 34.WVKQRPGQGLEWIG 35. KATLTADKSSSTAYMQLSSLTSDDSAVYFCAR 36. WGQGTTLTVSS 37.QVQL V QSGAEL KK PG A SVKVSCKAS 38. WVKQ A PGQGLEWIG 39. R ATLTADKS TSTAYM E LSSL R S E DSAVYCAR  40. WGQGTT V TVSS 41. GYAFTYYLIE 42.VINPGSGGTNYNEKFKG 43. NWMNFDY 44. QVQL V QSGAE VKK PG A SVKVSCKAS 45. WVR Q A PGQGLEWIG 46. R ATLTADKS T STAYM E LSSL R S E D T AVYFCAR 47. R ATI TADKS T STAYM E LSSL R S E D T AVYFCAR 48. RV T I TADKS T STAYM E LSSLR S E D T AVY Y CAR 49. MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVSVTPGESVSISC 50.WFLQRPGQSPQFLIY 51. GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC 52. FGGGTKLEIK 53.DIVMTQ TPL S L SVTPG QPA SISC 54. WFLQ K PGQSPQFLIY 55.GVPDRFSGSGSGTAFTL K ISRVEAEDVGVYYC 56. FGGGIK V EIK 57. RSSKSLLHSNGNTYLY58. RMSNLAS 59. MQHLEYPYT 60. GVPDRFSGSGSGT D FTL K ISRVEAEDVGVYYC 61. WY LQ K PGQSPQFLIY 62.Alternative cleavage sites can be between amino acids 21and 22 of preprotein compared to SEQ ID NO: 8APPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCT ISPY 63.Alternative cleavage sites can be between amino acids 27and 28 of preprotein compared to SEQ ID NO: 8QQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY 64.Human LOX mRNA sequenceATGCGCTTCGCCTGGACCGTGCTCCTGCTCGGGCCTTTGCAGCTCTGCGCGCTAGTGCACTGCGCCCCTCCCGCCGCCGGCCAACAGCAGCCCCCGCGCGAGCCGCCGGCGGCTCCGGGCGCCTGGCGCCAGCAGATCCAATGGGAGAACAACGGGCAGGTGTTCAGCTTGCTGAGCCTGGGCTCACAGTACCAGCCTCAGCGCCGCCGGGACCCGGGCGCCGCCGTCCCTGGTGCAGCCAACGCCTCCGCCCAGCAGCCCCGCACTCCGATCCTGCTGATCCGCGACAACCGCACCGCCGCGGCGCGAACGCGGACGGCCGGCTCATCTGGAGTCACCGCTGGCCGCCCCAGGCCCACCGCCCGTCACTGGTTCCAAGCTGGCTACTCGACATCTAGAGCCCGCGAAGCTGGCGCCTCGCGCGCGGAGAACCAGACAGCGCCGGGAGAAGTTCCTGCGCTCAGTAACCTGCGGCCGCCCAGCCGCGTGGACGGCATGGTGGGCGACGACCCTTACAACCCCTACAAGTACTCTGACGACAACCCTTATTACAACTACTACGATACTTATGAAAGGCCCAGACCTGGGGGCAGGTACCGGCCCGGATACGGCACTGGCTACTTCCAGTACGGTCTCCCAGACCTGGTGGCCGACCCCTACTACATCCAGGCGTCCACGTACGTGCAGAAGATGTCCATGTACAACCTGAGATGCGCGGCGGAGGAAAACTGTCTGGCCAGTACAGCATACAGGGCAGATGTCAGAGATTATGATCACAGGGTGCTGCTCAGATTTCCCCAAAGAGTGAAAAACCAAGGGACATCAGATTTCTTACCCAGCCGACCAAGATATTCCTGGGAATGGCACAGTTGTCATCAACATTACCACAGTATGGATGAGTTTAGCCACTATGACCTGCTTGATGCCAACACCCAGAGGAGAGTGGCTGAAGGCCACAAAGCAAGTTTCTGTCTTGAAGACACATCCTGTGACTATGGCTACCACAGGCGATTTGCATGTACTGCACACACACAGGGATTGAGTCCTGGCTGTTATGATACCTATGGTGCAGACATAGACTGCCAGTGGATTGATATTACAGATGTAAAACCTGGAAACTATATCCTAAAGGTCAGTGTAAACCCCAGCTACCTGGTTCCTGAATCTGACTATACCAACAATGTTGTGCGCTGTGACATTCGCTACACAGGACATCATGCGTATGCCTCAGGCTGCACAATTTCACCGTAT 65. Human LOX Protein SequenceMRFAWTVLLLGPLQLCALVHCAPPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRAREAGASRAENQTAPGEVPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY

While preferred embodiments of the present invention have been shown anddescribed herein, it will be clear to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. An isolated antibody or antigen binding fragment thereof, thatspecifically binds to an epitope having an amino acid sequence set forthas SEQ ID NO: 6.